CA2649688A1 - Methods for detecting a mycobacterium tuberculosis infection - Google Patents

Abstract

Methods for detecting an infection with Mycobacterium tuberculosis (Mtb) in a subject are disclosed. The methods include detecting the presence of CD8+ T cells that specifically recognize an Mtb polypeptide. The methods include in vitro assays for detecting the presence of CD8+ T cells in a biological sample, andin vivo assays that detect a delayed type hypersensitivity reaction. The methods can also' include detecting Mtb polypeptides and polynucleotides. Reagents for the detection of an Mtb infection are also disclosed.

Description

METHODS FOR OETECTING A MYCOBACTERIUM TUBERCULOSIS
INFECTION -PRIORITY CLAIM
.5 This application claims the benefit of U.S. Provisional Application No:
60/782,364, filed March 14, 2006, which is incorporated herein by reference.
STATEMENT OF-GOVERNMENT SUPPORT
This invention was made with United States government support pursuant to Grant No.=NIH-R01-AI48090 and Grant No. NIH NIAID HHSN266200400081C.
N01-AI-40081 from the National Institutes of Health; the United States government has certain rights in the invention. This invention was also made with support from the Department of Veterans Affairs.

1=5 FIELD
This application relates to the field of diagnosis, specifically to methods for detecting a Mycobacterium tuberculsQsis (Mtb) infection in a subject.

BACKGROUND
Mycobacteria are a genus of aerobic intracellular bacterial organisms that, upon infection bf a host, survive within endosomal compartrnents of monocytes and.
macrophages: Human mycobacterial diseases include tuberculosis (caused by M
tuberculosis), leprosy (caused by M leprae); Bairnsdale ulcers (caused by-M.
ulcerans), and various infections caused by M. marinum, M. kansasii, M.
scrofulaeeum, M sziulgai, M. xenopi, M fortuitum, M. chelonei, M.. haemophilum and M. intracellulare (see Wolinsky, E., Chapter 37 in Microbiology: Including Immunology and Molecular Genetics, 3rd Ed., Harper & Row, Philadelphia; 1980).

One third of the world's population harbors.llM tuberculosis and is at risk for developing tuberculosis (TB). In iminunocompromised patients, tuberculosis.is increasing at a riearly logarithmic rate, and multidrug resistant strains'are appearing.
In addit'ion, Mycobacterial strains which were previously'considered to be nonpathogenic strains (e.g., M. aviurn) have now become major killers of

-2-inuriunosuppressed AIDS paiients. Moreover, current Mycobacterial vaccines are either inadequate (such. as the BCG vaccine for M. tuberculosis) or'unavailable: (stich as for M.' leprae)' (Kaufinann, S:, Microbiol. Sci. 4:324-328, 1987; U.S.
Congress, Office of Technology Assessment, The Continuing Challenge of Tuberculosis, pp.
62-67, OTA-H-574, U.S. Government Printing Office, Washingtori, D.C:, 1993).
Inhibiting the spread otuberculosis requires effective vaccination arid accurate, early diagnosis of the disease. Currently, vaccination with live bacteria is the most efficient niethod for inducing protective immunity. The most common Mycobacterium employed for this purpose is Bacillus'Calmette-Guerin (BCG), an avirulent strain of Mycobacterium bovis. However; the safety and efficacy of'BCG
is a source of controversy and some countries, such as the United States, do not 'vaccinate the general public.
Diagnosis of tuberculosis is oommonly achieved using a skin test; which involves intradermaF exposure to tuberculin PPD (protein-purified derivative).
Antigen-specific T cell responses i:esult in measurable induration at the injection site by 48 to 72 hours -after injection, which indicates exposure-to Mycobacterial antigens. However, the sensitivity and specificity of this test are not ideal;
individuals vaccinated with BCG cannot be distinguished from infected individuals.
Accordingly, there is a need in the art =for improved diagnostic'methods for detecting tuberculosis.

SUMMARY
Methods for diagnosing an infection with Mycobacterium tuberculosis (Mtb) are disclosed herein. The methods can include detecting CD8+ T cells and/or CD4+
=. that specifically bind an Mtb polypeptide of interest. The=methods can also include detect'ing.a delayed type hypersensitivity reaction in a subject and/or can include detecting specific Mtb polypeptides and polynucleotides. The disclosed assays can be used individually or in combination. The Mycobacterium tuberculosis infection can-be a latent or act'ive infection. =
- In several embodiments, methods are provided for detecting Mycobacterium =.
tuberculosis -in a subject. These methods include. contacting a biological sample from the subject'comprising T cells, such as CD8+ T cells and/or CD4+ T cells, with

-3-one or more.Mycobacterium polypeptides, or an antigen presenting cell presenting the- one or more Mycobacteriuni polypeptides. The one or more Mycobacterium .
polypeptides include an amino acid sequence set forth as (a) one of the amirio'acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2,.SEQ ID NO: 3; SEQ ID NO:

4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID.NO: 9, SEQ ID. NO: 10, SEQ ID NO: 11 or SEQID_ NO: 12; or (b) , at least nine to twenty..
consecutive amino acids of at least one ofthe amino acid sequences set forth as SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ I.
NO: 6, SEQ ID NO: 7;'SEQ ID=NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:
1.0, SEQ ID NO: 11 or SEQ ID NO: 12, wherein the nine to twenty consecutive amirio acids specifically bind major histocompatibility complex (MHC) class I.
It is determined whether the T cells specifically recogn'ize the Mycobacterium polypeptide. = .
In additional embodiments, methods are provided for detecting.
15. Mycobacterium tuberculosis in a subject, wherein the methods include administering to the subject an effective amount of a Mycobacterium polypeptide into the skin, subcutaneously or intradermally. The Mycobacterium polypeptide includes an -. amino acid sequeiice set forth- as (a) one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ
ID 'NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:. 10, SEQ ID
'NO: 11 or SEQID NO: ' 1.2; or (b) - at least nine to twenty consecutive amino acids of at least one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO:
2, SEQ ID'NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ
ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ
. ID NO: 12, wherein the nine to twenty consecutive amino acids specifically bind major histocompatibility complex (MHC) class I. The presence of T cells that specifically recognize.the Mycobacterium polypeptide are detected in the subject:
In fitrther embodiments, methods are disclosed for detecting a Mycobacterium tuberculosis infection in a subject, wherein the methods include detecting the presence of a Mycobacterium polypeptide or a polynucleotide encoding the polypeptide in a sample from the subject. The Mycobacterium polypeptide includes an amino acid sequence set forth as one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ -ID NO: 3., SEQ ID NO:
4, SEQ ID NO: "5, SEQ ID "NO:. 6, SEQ ID NO: 7, SEQ. ID'NO: 8, SEQ ID NO: 9, SEQ.ID NO: 10,..SEQ ID NO: I I or SEQID NO: 12.
Additionally, reagents for the detection of a Iviycobacterium- infection in a subject are described.
'The foiegoing and other features and=advantages will become rriore appareint from the following detailed description of several embodiments; which:
proceeds with reference= to the accompanying figures. . : : 10 BRIEF DESCRIPTION OF THE
FIGURES

Figure 1 is two graphs showing the deterrnination of human effector cell frequencies ex vivo using the IFN-y ELISPOT assay. Magnetic bead-purified CD8 T cells were cultured with DC (20,000/well) either infected with Mtb (H37Rv, MOI
= = 50) or pulsed with peptide pool representing CFP 10 (5 g/ml each peptide;

1=5 mers overlap 11 aa) in an IFN- y ELISPOT assay. Each responding T cell population was tested in duplicate at four different cell concentrations. To determine the effector cell frequency of antigen-specific T cells, the average= number of spots per'well for each duplicate was plotted against the number of responder cells per well. Linear regression analysis was used to determine the. slope.
of the.
20 line, which represents the frequency of antigen-specific T cells. The assay was considered positive (reflecting the presence of a primed T cell response),,if the binomial probability for the number of spots was significantly different by experimental and control assays. . . " . =
Figure 2 is a set of 'graphs showing ex vivo CD8+ 'T cell frequencies to Mtb 25 antigens are associated with Mtb infection. As'described above (see Fig.
1), to determiiie ex vivo CD8+ T cell frequencies, autologous DC either infected with Mtb or pulsed with cognate peptide pools were incubated with CD8+ T cells =in an IFN-y ELISPOT. assay. Subjects without evidence for Mtb infection, those with LTBI, and those with active TB (culture confirmed pulmonary tuberculosis) were evaluated.
30 -"Mtb Infected" includes those with LTBI and active tuberculosis. P values are noted where P ==<0.05 (Wilcoxon/Kruskal-Wallis).

-5-Figures 3a to 3d are a set of digital images shovving the definition of Aiitigenic Specificity and HLA-Restriction (the Characterization of.T cell clone D466 D6). For the results shown in Figures 3a-3c, to Identify the*antigen-and minimal epitope recogriized by T cell' clone, D466 D6, T-cells (5000 cells/well) were incubated with autologous LCL (20,000/we11).and 5 g/ml 6f antigen.IFN- y was assessed by ELISPOT =after eighteen hours of co-culture.' For the results 'presented in Figure 3a, antigens consisted of peptide pools.representing known CD4+ antigens, made up of 15'amino acid (aa) peptides overlapping by 11 aa. :
For the results presented in Figure 3b, antigens consisted of individual 15 aa'CFP10 == peptides tliat together constitute the peptide pool. For the results presented in* Figure 3c, antigens consisted of individual nested CFP101_15 peptides (10 aa, 9 aa or 8 aa), :
used to'further map the epitope. For the results presented in Figure 3d, the restricting allele was identified using LCL (20,000/well) expressing. HLA
alleles matching D466 at one or two alleles, pulsed with CFP102_10 (5 g/ml) as APC.' After 2 hours, cells were washed and incubated with T-cells (500 cells/well) in an IFN- y ELISPOT assay. = ' _ =
Figure 4 is a line graph showing the confirmation of minimal epitope mapping of D466 D6. To confnm the minimal epitope, autologous LCL
(20,000/well) was pulsed with.peptide at the. concentration. indicated and co-cultured 26. with=T-cells (1000 cells/well). IFN-y was assessed by ELISPOT after eighteen hours co-culture. Each point represents the mean of duplicate determinations.
Figure 5 is a set of bar graphs showing the profiling of immunddominance pattern for CFP10. To determine the'effector cell= frequencies, autologous DC
(20,000 / well) were pulsed either with each individual 15-mer peptide (5 g/ml), .25 the peptide pool (PP; 5 g/each peptide) or the minimaY epitope (ME) determined from T cell clones derived from each donor-(D466:CFP10z_11; D480:CFP103_1 D48I:CFP1075_83i 5 g/ml), and tested against'250,000 magnetic bead purified CD8+

T cells. IFN-y release was assessed by ELISPOT after-eighteen hours of co-culture.
Each point represents the mean of duplicate determinations. .
.30 Fig. 6 is a set of graphs summarizing the =minimal. epitope mapping data.
To determine the miniinal epitope, autologous LCL-(20,000/well) was pulsed with peptide at the concentration indicated and co-cultured with T-cells (1000 ce11s/well)-.

: -6-IFN-,y was a'ssessed by ELISPOT after eighteen hours co-culture.' Each point represents the mean'of duplicate determinations.
Figure.7 is a line graph showing the mapping of Minirnai Epitope for D504 Clones. ' Ta determine the minimal epitope, autologous LCL (20,000/well) was co=
cultured with T-cell clones (1,000cells/well) and:the peptide,at the concentration indicated. IFN=y was assessed by ELISPOT after eighteen hours co-culture. Each point represents the mean of duplicate determinations. .: SEQUENCE LISTING

The nucleic and:amino acid sequences listed in the.accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amirio acids, as defined in 37 C.F.R. 1.822.= Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as iincluded by any reference to. the displayed strand. In the accompanying sequence . listing: =, . . -SEQ Il) NOs: 1-12 are the amino acid sequence of Mtb polypeptides.
SEQ ID NOs: 13-14 are amino acids of Mtb peptides.
SEQ ID NOs:' 15-25 are the nucleic acid sequences of polynucleotides encoding the Mtb polypeptides. -= SEQ ID NOs: 26-38 are the amino acid sequences of specific Mtb epitopes.
DETAILED DESCRIPTION

Methods for detecting an infection with Mycob'aeterium tuberculosis in a =, subject are disclosed. The methods include detecting the presence of T
cells, such as but not limited to CD8+ T cells, that specifically-recognize aMyeobacterium tuberculosis (Mtb) polypeptide. The:methods include in vitro assays-for detecting the presence of CD8+7 cells in a biological sample, and in vivo assays that detect a delayed type hypersensitivity reaction. The rnethods can also include detecting Mtb polypeptides and polynucleotides: Reagents for the. detection of an Mtb infection are also disclosed. .

Terms Unless -otherwise noted, technical terms are used accordirig to cornven=tional usage. Definitions of common terms in molecular=biology may be found in ' Benjamin Lewin, Genes V, published by Oxford University.Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, piiblished by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.1Vleyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference;
published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). - : :
In order to facilitate review of the various embodiments of.this disclosure, : the following explanations of specific terms, are provided:
Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which.
antigeri ' 'is adsorbed; or wate=r-in-oil emulsion in which antigen solution is emulsified in -mineral oil (Freun:d incomplete adjuvant), sometimes with the inclusion of killed .15 mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits =degradation of antigen and/or=causes influx of macrophages).
Irnmunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as acijuvants (for example see U.S. Patent No. 6,194,388; U.S. Patent No. 6,207,646; U.S.
Patent No. 6,214;806; U.S. Patent No. 6;218,371; U.S. Patent No. 6,239,116; U.S.
Patent No. 6,339,068; T.J.S. Patent No. 6,406,705; and U.S. Patent No. 6,429,199)..
Adjuvants include biological molecules (a "biological adjuvant"), such as costimulatory molecules. Exemplary adjuvants include:IL-2, RANTES, GM-CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1; B7-2, OX-40L and 41= BBL
Amplification: Of a nucleic acid molecule (e.g., a DNA or RNA molecule) .' -25 refers to use of a technique that increases the number of copies of a nucleic acid molecule in a specimen. An example of amplification is the polymerase chainreaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to a nucleic acid-template in the sample. The primers are extended.
under suitable'conditions, dissociated frorri the template, and then re-annealed;
exterided, and dissociated to amplify the nuniber of copies of the inucleic acid. The product of amplification can be characterized by electrophoresis, restriction -8- .
endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing using standard techniques. Other examples of amplification ==
include strand displacement amplification, as disclosed in U.S.=Patent No.
5,744,3.11,; transcription-free isothermal amplification,.as disclosed.in U.S.
Patent No: 6,033,881; repair chain reaction amplificatiorl, as disclosed in WO
90/01069;
ligase chain reaction amplification, as disclosed in EP-A-320 308; gap filling ligase -chain reaction amplification, as disclosed in U.S: Patent-No. 5,427;930; and NASBATM RNA transcription-free amplification, as disclosed in U.S. Patent No.

6,025,134. . 10 = Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. =An antigen reacts with'the *products of specific' humoral or cellular immunity, including those induced by heterologous immunogens. The.term="antigen" includes all related antigenic epitopes.
"Epitope"
.' T5= or "antigenic determinant" refers to a site on an antigen to which B
and/or T cells -' respond. In one enibodiment, T cells respond to the 'epitope, when the epitope is =.presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids= or noncontiguous amino acids juxtaposed by tertiary foldirig of a protein. Epitopes formed from contiguous amino acids are typically retained on 20 exposure to denaturing=solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3,and more usually,-at least 5, about 9, or about 8-10 amino acids in=a unique -spatial conformation. Methods of determining spatial conformatioil of epitopes include, forexample, x-ray crystallography and 2-dimensional nuclear=magnetic =
25 resonance.
An antigen can be a tissue-specific antigen, or a disease-specific antigen.
These terms=are not exclusive, as a tissue-specific antigen can also be a=disease specific antigen. A tissue-specific antigen is expressed in a limited number of tissues, such as a single tissue. A tissue specific antigen may be expressed by more =
30 tha n one tissue, such as, but not limited to, an antigen that is expressed in more than one reproductive tissue, such as in both prostate and uterine tissue. 'A.
disease- ==
specific antigen is expressed coincidentally with a= disease process. Specific non--9- . . -limitin=g examples of a disease-specific antigen are an aritigen whose expression -correlates with, or.is-predictive of, tuberculosis. A disease-specific antigen can=be an antigen recognized by Tcells 'or B cells. :. "
Antibody:' Iinmunoglobulin inolecules and immunologically active portions 5=' of inununoglobulin rnolecules, i.e., molecules -that contain an antigen binding site that specifically binds (iminunoreacts with) an antigen, such as anMtb *polypeptide.
A naturally occurring antibody (e.g.,, IgG,. IgM, IgD) includes four.
polypeptide chains, two heavy (H) chain's and two light (L) chains interconnected by disulfide bonds. However, it has been shown that the antigen-binding function of an 10. antibody can be performed by fragments of a naturally occurring antibody.
Thus, these antigen-binding fragments are also intended to be designated by the term "aritibody." Specific, non-limiting exarnples of binding fragments encompassed within the term antibody include (i) a Fab fragment consisting of the VL, VH, CL and CHI domains; (ii) an Fd= fragnient consisting of the VH and CuI domains; (iii) an Fv 15 fragment consisting of the VL and VH domains of a sirigle arm of an antibody, (iv) a dAb fragment (Ward et al., Nature 341:544-546, 1.989) which consists of a VH
domain; (v) an isolated complementarity determining region (CDR); and (vi) "a F(ab')2 fragmerit, a bivalent fragment comprising two Fab fragments linked by a = . .
disulfide bridge at the hinge region.
.20 Imniunoglobulins and certain variants thereof are khown and many have been prepared in recombinant cell culture (e.g., see U.S. Patent No.
4,745,055; U.S.
Paterit No. 4;444,487; WO 88/03565; EP 25.6,654; EP 120,694;: EP 125,023;
Faoulkner et al., Nature 298:286, 1982; Morrison,.J.. Immunol. 123:793, 1979;
Mor=rison et al., Ann=Rev. Immuno12:239,.1984).
25 Animal: Living mi.ilti-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and - veterinary subjects. .
'Antibody: Immunoglobulin molecules and immunologically active portions 30 of immunoglobulin molecules, i.e., molecules that contain ari antigen binding site that specifically binds (immunoreacts with) an =antigen. =

'A naturally occurring antibody (e.g., IgG, IgM, IgD) includes four.
polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disiulfide bonds. However, it has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody:.
Thus, these, antigen-binding fragrnents are also intended to be designated by the feriii "antibody." Specific, =non-limiting examples of binding fragments encompassed within the'term antibody include-(i) a Fab fragment consisting of the VL, VH, CL and CHI domains; '(ii) an Fa fragment consisting of the VH and CHI domairis; (iii) anFv =
fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a 1'0 dAb fragment (Ward et al., Nature 341:544-546, 1989) which consists of a VH
domain; (v) an isolated complimentarity determining region (CDR); and (vi) a F(ab')Z fragment, a bivalent fragment comprising two Fab fragments linked by a-disulfide bridge at the hinge region.
Immunoglobulins and ceirtain variants thereof are known and many have been prepared in-recombinant cell culture (e.g.,. see U.S.' Patent No.
4,745,055.; U.S.
Patent No. 4,444,487; .WO 88/03565; EP 256,654; EP 120,694; EP 125,023;
Faoulkner et al., Nature 298:286, 1982; Morrison, J. Immunol. 123:793, 1979;
Morrison et al., Ann Rev. Immunol 2:239, 1984). = '.
Antigen presenting cell (APC): A cell that can present an antigen to T cell, such that the T cells are activated. Dendritic cells are the principle antigen presentiing cells (APCs) involved in primary immune responses. Their major function is to obtain antigen in tissues, migrate to lymphoid organs and present. the antigen in order to activate T cells. .=
When an appropriate maturational cue is received, dendritic cells are signaled to undergo rapid morphological and physiological changes that facilitate the initiation and development of immune responses. Among these are the up-regulation of molecules involved in antigen presentation; 'production of pro-inflammatory cytokines, including IL-12, key to the generation of Thl responses;
and secretion of chemokines that help to drive differentiation, expansiori, and migration of surrounding naive Th cells. Collectively; these up-regulated molecules facilitate the ability of dendritic cells to coordinate the activation and effector .-11-function.of other surrounding lymphocytes that ultimately provide protection for the host.
eDNA (complementary DNA): A piece of DNA lackinginternal, non-coding segments (introris) and regulatory sequences that determine transcription.
cDNA,is synthesized in the laboratory by reverse transcription from-messenger RNA
extracted from cells.
CD4: Cluster of differentiation factor 4, a T cell surface protein that mediates interaction with the MHC Class II molecule: . CD4 also ser'ves =as the primary receptor site for HIV on T cells during HIV infection. Cells that express CD4 are often helper T cells.
CD8: Cluster*of differentiation factor 8, a T cell su=rface protein that mediates interaction with the MHC Class I molecule. Cells that express CD8 are 6ften cytotoxic T cells. "CD8+ T cell mediated immunity" is an imrriune response irnplerriented by presentation of antigens to CD8+ Tcells. . =
= cDNA (complementary DNA): A piece of DNA lacking internal, non-coding seginents (introns) and regulatory sequences that determine transcription.
cDNA is synthesized in the laboratory by reverse transcription from messenger RNA
extracted from cells. =
Conservative variants: "Conservative" amino acid substitutions are those '20 substitutions that do not substantially affect or decrease an activity or antigenicity of the Mycobacterium polypeptide. Specific, non=limiting examples'of a conservative substitution.include the following examples:

=12-Original Residue Conservative Substitutions Ala . . Ser Arg Lys Asn Gln, His Asp = . . Glu Cys . . Ser Gln = Asn .10 . Glu = . , Asp His . : Asn;= Gln .
Ile ' . - Leu, Val Leu = ' Ile; Val Lys . . Arg; Gln; Glu Met I,eu; Ile Phe Met; Leu; Tyr Ser Thr Thr . Ser Trp Tyr -Tyr Trp; Phe Val Ile; Leu The term 'conservative variation also. includes the use of a substituted amino acid in place of ari unsubstituted parent amino acid, provided that antibodies raised . 25 to the substituted polypeptide also immunoreact with the unsubstituted polypeptide, or that an immune response cari be generated against the substituted polypeptide that is similar to the immune response against and unsubstituted polypeptide, such a Mycobacteriurn antigen. Thus, in one embodiment, non-conservative substitutions, are those that reduce an activity or antigenicity.

30, Consists Essentially Of/Consists Of: With regard to a polypeptide, a polypeptide that consists essentially of a specified amino acid.sequence if=it does not include any additional amino acid.residues. However, the polypeptide can=include additional non-peptide components, such as labels (for example, fluorescent, radioactive, or solid particle labels), sugars or lipids. A polypeptide that consists of 35 a specified:amino acid sequence does not include any additional amino acid -residues, nor does it include additional non-peptide components, such as lipids, sugars or labels. ' .

Contacting: The process of incubating oiie agent in the presence of another.
Thus, when= a cell is contacted with an agent; tlie cell is inciibated with the agent for ' a sufficient period of time for the agent and the cell to=interact.
Costimulatory molecule: Although engagement of the TCR with peptide-MHC delivers one signal to the T cell, this signal alone canbe insufficient to =
activate the T cell. Costimulatory molecules are molecules that, when bound to-their ligand, deliver a second signal required for the T cell to become activated.
The most well-kriown costimulatory inolecule on the T cell is CD28, which binds to either B7-1(also called CD80) or B7-2 (also known as CD86). An additional costimulatory molecule is B7-3. Accessory molecules that also provide a secoind signal for the activation of T cells include intracellular adhesioin molecule (ICAM-1 and ICAM-2), leukocyte function associated antigen (LFA-1, LFA-2 and LFA-3). Integrins and tumor.necrosis factor (TNF) superfamily members can also serve as co-stimulatory = molecules. . . = .

Cytokine: Proteins made by cells that affect the behavior of other cells;
such as lymphocytes. In one embodiment, a cytokine is a chemokine, a molecule that affects cellular trafficking. = Specific, rion-limiting examples of cytokines include the interleukins (IL-2, IL-4, IL-6, IL-10, IL-21, etc.), and interferon (IFN)--y:
Degenerate variant: A polynucleotide. encoding an epitbpe of an Mtb=-= polypeptide that includes a sequence that is deg'enerate as a result of the genetic code. = There are 20 natural amino acids, most of which' are specified-by more than one'codon. Therefore,.all degenerate nucleotide sequences.are included in this disclosure as long as the amino acid sequence of the Mtb polypeptide encoded by the. inucleotide sequence is unchanged.
Dendritic cell (DC): Dendritic cells are the principle antigen presenting cells (APCs) involved in primary immune responses. Dendritic cells-include plasmacytoid dendritic cells and myeloid deindritic cells. Their major function is to obtain antigen in tissues, migrate to lymphoid organs and present the antigen in order to activate T cells. Immature dendritic cells originate in the bone marrow and 30. reside in the.peripl=iery as immature cells. . .= -Diagnostic: Identifying the presence or nature=of a pathologic condition,, such as, but not limited to, tuberculosis. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a,diagnostic assay is the peicentage of diseased.individuals.who test positive (percent of true positives). The "specificity" of a diagnostic'assay is I minus the false positive rate, where the false positive rate is defined as the- proportion of those =without the diseas'e' who' test positive. While a particular diagnostic method may not provide a definitive ' diagnosis of a condition, it'suff ces if the inethod provides a positive indication that aids in diagnosis. "Prognostic" means predicting the probability of development (for example, 'seveiity) of a pathologic condition, such as=tubercuiosis. .
Displaying: The process of localizing a peptide:antigen complex, or a 10' peptide, on the outer surface of a cell where the peptide:antigen complex or peptide is accessible to a second cell, molecules displayed by a second cell, or soluble factors. A peptide, or-a peptide:antigen complex, is "displayed" by a*cell when it is present on the outer surface of the cell and is accessible to a second cell, to molecules displayed by the second cell, or to soluble factors. -15 . Epitope:
An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule=that are antigenic, i.e. that elicit a specific inimune response. An antibody specifically binds= a partioular antigenic epitope ori a polypeptide, such a Mycobacterium polypeptide. ' . ' Expression Control Sequences: Nucleic acid sequences that regulate the ' 20 expression of a heterologous nucleic acid sequence to which -it, is operatively linked.
Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as ..
appropriate, -translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhan cers, transcription terminators, a 25. start.codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at.a minimum, components whose presence can influence expression, and .can also include additional components whose presence is advantageous, for .
30 example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

A promoter is a minimal sequence sufficient to direct transcription. Also :' included are those promoter elements which are sufficient to render prombter-dependent gene expression controllable for cell-type specific; tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the.gene. Both constitutive and inducible promoters, are.included (see =
e.g , Bitter et aL, Methods in Enzym6logy'153:516-544, 1987). For.=example, when-cloning in bacterial systems, inducible prorrioters such as pL 'of liacteriophage lambda , plac, ptrp, ptaa (ptrp-lac hybrid promoter) and the like may be used.
In.one embodiment, when cloning in mammalian cell systems, promoters derived from the - genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terrrriinal repeat; the adenovirus late promoter; fihe vaccinia=virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences. In one embodiment, the promoter is a cytomegalovirus promoter.
Fractionating: Subjecting a sample to conditions or procedures which.
separate the components of the sample based on physical or cliemical properties such as, but not limited to, size, 'charge, solubility, or composition.:
Example of fractionation procedures include, but are not limited to, selective precipitation, organic extraction, size exclusion dialysis or chromatography, such as ion exchange chromatography. In one embodiment, a fraction is a soluble extract or an organic extract of an organism, such as a Mycobacterium.
Functionally. Equivalent: Sequence alterations, such as in an.epitope of an antigen, that yield the same results as described herein. Such sequence alterations can include, but are not limited to, conservative substitutions, deletions, mutations;
' frameshifts, and insertions:
Heterologous: Originating from separate genetic sources or species. A
polypeptide that is heterologous to an Mtb polypeptide originates from a nucleic acid that does not encode the Mtb polypeptide. In,one specific, non-limiting example, a polypeptide comprising nine consecutive amina acids from an Mtb polypeptide, or at most 20 consecutive amino acids.'from the Mtb polypeptide, and a heterologous amino acid sequence includes a0-galactosidase, a maltose binding -16- =protein, and albuinin,=liepatitis B surface antigen,'or ain inunurioglobtilin amiiio acid sequence. -Generally, an=antibody.that specifically binds to.a protein of interest.will not specifically bind to a= heterologous protein. .: :. =' Host cells:. Cells in which a vector can be propagated and its DNA
5. expressed. = The cell may be prokaryotic or eukaryotic=. The cell caii be riiammalian, such as a human cell. The term also -includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the' parental cell sirice there may be mutations that occur during replication. 'However, such progeny are included when the term "host.cell' is used. 10. HumaiT Leukocyte Antigen (HLA): A genetic designation of the human inajor histocompatibility complex (MHC). Individual loci are designated by uppercase letters, as in HLA-E, and alleles are designated by numbers, as in HLA-A*0201. The three main MHC 'class I genes are called HLA-A, HLA-B, and.HLA-C. However, there are many genes that encode 02 microglobulin-as'sociated cell=
15 surface molecules that are linked to the MHC class I genes. The expression of these genes is variable, both in the tissue distribution and=the amount expressed on=cells;
these.genes have been termed the MHC class IB -genes.
Immune response: A response of a cell of the immune system,. such as a B
cell, natural killer cell, or a'T cell, to =a stimulus. In oine embodiment, the -response is 20 specific for a paiticular antigen (an "antigen-specific response"). In one .
embodimerit, an immune response is a T cell response, such as a Thl; Th2, or Th3 = response. In another embodiment, an immune response is a response of a suppressor Tcell.
Immunogenic peptide: A peptide which comprises an allele-specific motif 25 or other sequence such that the peptide will bind an MHC molecule and iriduce a T
.cell response, such as a CD8' T cell response, or a B-cell response (such as antibody production) against the antigen from which the immunogenic peptide is derived.
In one embodiment, immunogenic peptides are identified using.sequence motifs or other methods, such as neural net or polynomial determinations, known in, 30 the art. Typically, algorithms are used to determine the "binding threshold" of ' peptides to select those with scores that give them a high probability, of binding at a certain affinity aind will be immunogenic. The algorithms are based either on the effects on MHC binding of a particular amino acid at a particular position, the effects on antibody biriding of a particular amino acid at a particular position, or the effects on binding of a particular.substitution in a motif-containing peptide:
Within the context of an immunogenic peptide, a"conserved residue" is one which appears in a'significantly.higher frequency than would-be expec'ted by random distribixtion.at a particular positioin in a peptide. In one embodiment,=a conserved residue is= one where the MHC. structure may provide a contact point with the immunogenic .
peptide.. .
Immunogenic peptides can also be identified by measuring their binding to a = specific MHCprotein and by their ability to stimulate CD4 and%or CD8 when presented in the context of the MHC protein. In one example, an immunogenic =` Mtb peptide" is a series of contiguous amino acid residues from the Mtb protein generally between 9 and 20 amino acids in length, such as about 8 to 11 residues in length. Specific immunogenic polypeptides are disclosed herein that are 9 or amino acid residues in length, or at most 12 amino acids in length. .
Generally, immunogenic Mtb polypeptides can be used, to induce an immune response in a subject, such as a B cell response or a T cell response. In. one example, =
an immunogenic Mtb polypeptide, when bound to a Major Iiistocompatibility Complex Class I molecule, activates CD8+ T cells, such as cytotoxic T
lyinphocytes (CTLs) against.Mtb.. Induction of CTLs using synthetic peptides and CTL
cytotoxicity assays known in the art, see U.S. Patent 5,662,907, which is incorporated herein by reference. In one example, an immunogenic peptide includes an allele-specific motif or other sequence such that the peptide will= bind ah MHC
molecule and induce a CD8+response against the antigen from which the irnmu.nogenic peptide is derived. A CD8+ T cell that specifically recognizes an Mtb polypeptide is activated, proliferates, and/or secretes cytokines in response to that =
specific polypeptide, and not to other, non-related polypeptides.
Immunogenic composition: A composition comprising* an immunogenic Mtb polypeptide or a nucleic acid encoding the immunogenic Mtb polypeptide that induces =a measurable T response. against Mtb, such as a.CD8* T cell response, or induces a measurable. B cell .response (such as production of antibodies that specifically bind an Mtb polypeptid'e). For.in vitro use, the immunogenic composition =can consist of the isolated nucleic acid, vector including the nucleic acid/or immunogenic =peptide. For in vivo= use, the immunogenic composition wi-ll typically comprise the nucleic acid, vector including the nucleic acid,'anci or immunogeriic polypeptide, in pharmaceutically acceptable carriers, and/or other agents. An immunogenic composition can optionally include an adjuvant, a costimulatory molecule, or a nucleic acid encoding a costimulatory molecule..
An .
Mtb polypeptide, or nucleic acid encoding the polypeptide, can=be readily tested for its ability to induce a CD8+=T cell response. ::
Inhibiting or treating a disease: Inhibiting a, disease, such as tuberculosis, 'refers to inhibiting the full development of a disease. In several examples, inhibiting a disease=refers to lessening symptoms of a tuberculosis. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to the disease, such as tuberculosis.
Interferon gamma (y):. IFN-y is a dimeric protein with subunits of 146 amino acids. The protein is glycosylated at two sites, and the pI is 8.3-8.5.
IFN-y is synthesized as a precursor protein of 166 amino acids including a secretory.signal sequence of 23 amino acids. Two moleculai= forms of the biologically active protein.
of 20 and 25 kDa have been described.. Both of thein are glycosylated=at position 25. The 25 kDa fonn is also glycosylated=at position 97. The- observed differences of natural IFN-y with respect to molecular mass and charge are due to variable glycosylation patterns. 40-60 kDa forms observed under non-denaturing conditions are dimers. and tetramers of IFN-y. The humangene has a length of apprbximately'6 kb. It contains four exons and maps to chromosome 12q24.1.
IFN-y can be .detected by sensitive immunoassays, such as an ELSA test that allows detection of individual tells producing IFN-y. Miriute amounts of IFN-y can be detected indirectly by measuring YFN-induced proteins such as Mx protein..
The induction of the synthesis of IP-10 has been.used also to measure IFN-y .
concentrations. In addition, bioassays can be used to detect IFN-y, such as an assay that employs induction of indoleamine 2,3-dioxygenase. activity in 2D9 cells.
The ~
production of IFN-y can be used to assess T cell activation, such as activation of a=T
cell by an HLA-E presented Mycobaeterium antigen.

= Isolated: An `isolated" nucleic acid=has been, substantiall-y separated or purified away froni other nucleic acid sequences 'in the cell. of the organisni in which the nucleic acid naturally occurs; i.e.; other chromosomal and extrachromosomal DNA and RNA. The term "isolated" thus encompasses nucleic acids.purified by 5. standard, nucleic acid purification methods. The term also embraces=
riucleic acids prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
Label: A detectable compound or composition'that is coinjugated directly or indirectly to another molecule to facilitate.detection of that molecule.
Specif c., non-. limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive,isotopes.
Linker sequence: A linker sequence is an amino acid.sequence that .
covalently links two polypeptide domains. Linker sequences can be included in the 'between the Mtb epitopes disclosed herein to provide rotational freedom to the linked polypeptide domains and thereby to promote proper domain folding and presentation to the MHC. By way of example, in a recombinant polypeptide comprising two Mtb domains, linker sequences can be provided between them, "such as a polypeptide comprising Mtb polypeptide-linker-Mtb polypeptide. Linker sequences, which are generally between 2 and 25 'aniino acids in length, are well known in the =art arnd include, but are not limited to, the glycine(4)-seririe spacer (GGGCiS, x3) described by Chaudhary et a1.,11~ture 339:394-397, 1989.
Lymphocytes: A type of white blood cell that is involved in the imrimune defenses of the body. There are two main types of lymphocytes: B cells and T
cells.
Mammal: This=term includes both human and non-human mammals.
Similarly, the term "patient" or "subject" includes both human andveterinary =
subjects. . . ' Mycobacteria: A genus of aerobic intracellular bacterial organisms.. Upon invasion of a host, these organisms survive within endosomal compartments of monocytes and macrophages. Human mycobacterial diseases include tuberculosis (cause by M. tuberculo.sis), Leprosy (caused by, M. leprae), Baimsdale ulcers (caused by M. ulcerans), and other infections that'can be caused by M.
marinum, M.
kansasii, M. scrofulaceum, M szulgai, .~l~f. xenopi, M fortuitum, M.
haemophilum, M. chelonei,'and M. intracelluare. MycoBacterium strains that were previously considered to be nonpathogenic (such as M. avium) are:alao now known to be major killers of immunosuppressed AIDS patients.
The major response to mycobacteria involves cell mediated hypersensitivity (DTH) reactions with T cells and macrophages playing major roles in the intracellular killing and walling off (or containing) of the organism (granuloma formation). A major T cell response involves CD4+ lymphocytes that recognize myocbacterial heat shock proteins and immunodominant aritigens.
Operably linked: A first nucleic acid sequence is operably linked'with a second nucleic acid sequence when the first nucleic acid sequence is=placed in a functional relationship with the second nucleic acid sequence. For instance., a promoter is operably linked to a coding sequence-if the promoter effects the transcription or expression of the coding sequerice. Generally, operably linked DNA
Sequences are contiguous and, where necessary to join two proteiri coding'regions, the. open reading frames -are aligned. . .= "
ORF (open reading frame): A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually.
translatable into a polypeptide. . Peptide Modifications: Mycobacterium polypeptides include synthetic 20: embodiments of peptides described herein. In addition,, analogues (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting=withthe disclosed peptide sequences) and variants (homologs) of these proteins can be utilized in the methods described herein. Each polypeptide of the invention is comprised of a sequence of amino acids, which inay be either L-and/or D- amino acids, naturally occurring and otherwise. ' Peptides may be modified by a variety. of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other. desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the forrn of a salt of a pharmaceutically-acceptable cation or esterified to form a C1-C16 ester; or =
converted to an amide of formula NR1 R2 wherein Rl and RZ= are each independently H or CI-C16 alkyl, or combined to form a heterocyclic.ring, such as a 5- or 6--21- . =
=
'membered ring. Amino groups of the peptide, whether amino-terminal or side.
chairi, may be in the form of a pharmaceuiically-acceptable acid addition salt, suclh' as the HCI, HBr, acetic, benzoic; toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to CI -.C16 alkyl or dialkyl amino or further converted- to an =amide.: :
Hydroxyl groiups of the peptide side chains may be converted to CI-CI6 alkoxy or to a Ct-C1 ester using well-iecogn-ized techniques. Phenyl and phenolic rings of-the peptide. side chains may be substituted with one'or more'halogen aloms, such as fluorine, chlorine, bromine or iodine, or with C1-C16 alkyl, CI-C16 alkoxy, 10. carboxylic acids and esters. thereof, or amides of such oarboxylic acids.
Methylene groups of the peptide side chains canbe extended to homologous C2-C4 alkylenes.
Thiols can be protected with any one of a riumbeir of well-recognized =protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic'structu.res into the peptides of this inventiori to select =
and provide conformational constraints to the structure that result in enhanced stability. , Peptidomimetic and organomirnetic embodiments, are envisioned, wheireby the three-dimensional arrangement.of the chemical constituents of such peptido-and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting iin such peptido- and organomimetics of a Mycobacterium polypeptide having measurable or enhanced ability to generate an immune response. For computer modeling applications, a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed .25 to fifeach pharmacophore.with current computer modeling=software (using computer assisted drug design or CADD). See Walters, "Compiuter-Assisted Modeling of Drugs", in Klegerman & Groves, eds., 1993, Pharmaceutical.
Biotechnology, Interphaixn Press: Buffalo Grove; IL,.pp. 165-174 and Principles of Pharmircology Munson (ed.) 1995, Ch. 1.02, for descriptions of techniques used in CADD. Also included are=mimetics prepared using such techniques.

-22-.. ... _. . =
Phar-maceutical agent or drug: A chemical coinpound or 'corriposition capable of inducing a desired therapeutic or prophylactic effect when properly 'administered to a subject.
Pha'rmaceutically acceptable carriers:. The -pharmaceutically acceptable carriers..useful with the polypeptides and nucleic acids described'herein are conventional. Remington's Pharmaceutical Sciences; by E. W. Martin; Mack Publishing Co., Easton, PA, 15th Edition (1975); describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed.
.' . In general, the nature of the carrier will depend on the particular mode of adrninistration-being employed. For instance, parenteral formulations usually' comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a-vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic.solid carriers can include, for example, pharmaceutical' grades of mannitol, lactose, starch, or magnesium.stearate. In addition to biologically-neutral carriers, pharmaceutical =
compositions to be.administered can contain minor amounts of non-toxic auxiliary =' substances, such as wetting or emulsifying agents, preservatives, and pH
buffeiring ' agents and the like, fo =r example sodium acetate or sorbitan moriolaurate.=
Polynucleotidei A linear nucleotide sequence, including sequences of greater than 100 nucleotide bases in length.
Polypeptide: Any chain of amino acids, regardless.of length or post-translational modification (e.g., glycosylation or phosphorylation).
A"peptidex'=is a 25. chain of amino acids that is less than 100 amino acids in length. = In-orie embodiment, a `peptide" is a portion of a polypeptide, sucli'as at.about 10, 20, 30, 40, 50, or 100 contiguous amino acids of a polypeptide that is greater=than 1:00 amino acids in length.. = ' ' =
Portion of a nucleic acid sequence: A.t least 10, 20; 30 or 40 contiguous nucleotides of the relevant sequence, such as:a sequence encoding an antigen.
In some instances it would be advantageous to use a portion consisting of=50 or more nucleotides. For instance, when describing a portion of an antigen (such as an antigenic.epitope); it miay be advantageous to remove a porti.on of.the relevant sequence comprisirig at least '10, 20, 30, 40 br 50 nucleotides up to a length.
Probes and primers: Nucleic acid probes =and primers 'may readily be prepaxed based on the nucleic acids provided by this invention. A probe comprises 5= an isolated nucleic acid attached to a detectable label or reporter inolecule: Typical labels include radioactive isofopes, ligands, chemiluminescent, agents, and enzyrnes.
Methods for labeling and guidance in the clioice of labels'appropriate for 'varioiis purposes are discussed,=e.g., in Sambrook ef al: (1989) axidAusubel et al.
(1987).
Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length. Primers may be arinealed to.a complementary target DNA strand'by nucleic acid hybridization to form a hybrid between the primer and =the target DNA stratnd, and then extended along the target DNA strand by'a DNA
polymerase enzyme. Primer pairs can be used for amplification of a nucleic aaid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
Methods for preparing and using probes and primers are described,'for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol: 1-3, ed.
Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Current Protocols in Molectilar Biology,. ed: Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1987 (with periodic updates). PCR
= primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, (D
1991,'Whitehead Institute for Biomedical Research, Cambridge, MA).
Preventing or treating a disease: "Preventing" a disease refers to inhibiting the full development of a disease, for example in a person who is knowri to be at risk of infection with M. tuberculosis, or M. leprae. An example of a person with a known predisposition is someone living with a person diagnosed with tuberculosis, health care professionals, or someone the family, or who has been exposed-to M tuberculosis. "Treatment" refers to a therapeutic intervention that ameliorates a sign or sy.mptom of a disease or pathologioal.condition, such as tuberculosis, after it has begun to develop. == .

Promoter: A promoter is an ariay of nucieic=acid control.sequences which direct transcription of a nucleic acid. A prornoter includes necessary nucleic acid sequences.near the start site of transcription, such as, iri -the. case of a polyrrierase II
type promoter, a. TATA element. A promoter also optionally includes distal.
S. =enhancer or repressor elements which can be located as much as several thousand base pairs'from the start site.of transcriptiori. = The promoter can be a constitutive or .
an inducible promoter. A specific, non-limiting example of a proinoter is =the HCMV IE promoter. :
Purified: The term purified does not requite absolute purity; rather, it is intended as a relative term. Thus, for example, a purified antigen preparation is one in which the'antigen is more pure than the protein in its originating environment =
within a cell. A preparation of an antigen is typically purified such that the aiitigen .
represents at least 50% of the total protein content of the preparation.
However, more highly purified preparations, may be required for certain applications.
For example, for such applications, preparations in which the antigen comprises at=least 75% or at least 90% of the total protein content may be employed.
Recombinant: A recombinant nucleic acid or polypeptide is oine. that has a sequence that is not naturally occurring or has a sequence'that i-s rnade by an'.
artificial combination of two or more otherwise separated segments of.sequence.
This artificial corimbination-is often accomplished by cherriical synthesis or, more commonly, by tlie artificial manipulation of isolated.segments of nucleic acids, e.g., by genetic engineering techniques. . =
Sequence identity: The similarity between amino acid sequerices is expressed in terms of the sirriilarity between the sequences, otherwise referred to as sequence-identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Variarits of-antigen polypeptides will possess arelatively high degree of sequence identity when aligned using standard methods.
Methods of aligriment of sequences for comparison are well known in the art.
Altschul et al. (1994) preserits a detailed consideration of sequence alignment methods and hornology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and: on the Internet, for use in.connection with the sequence analysis programs blastp, blastn, =
blastx, tblastn and tblastx. 'It can be =accessed 'at theNCBI website. A
description of how to .deternnine sequence identity using this program is available at the =NCBI

website, as are the default parameters. Variants of antigenic polypeptides, such -as a Mycobaeterium polypeptide, are typically characterized by possession of at least. 50% sequence identity counted over the full length alignment with the amino =acid sequenae of a native arYtigen' sequence using the NCBI Blast 2.0, gapped blastp set to default paraiimeters.
= 10 Proteins=with even. greater simitarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at.least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at =least 90% or at least 95%
sequence'.".
identity. When less than the entire sequence is being compared for sequence' identity, variants will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85%
or-at least 90% or 95% depending on their similarity to the reference sequence:
Methods for determining sequence identity over such short windows are.described at the NCBI website. Variants of MHC domain polypeptides also retain the biological activity of the native polypeptide. For the purposes of this inveiation, that"activity is conveniently assessed by incorporating the variant domain in the =appropriate (31a1 or a1a2 polypeptide. and determining the ability of the resulting polypeptide to inhibit antigen specific T-cell proliferation in vitro, or to induce T
suppressor cells or the expression of IL-10 as described in detail below. . .
Therapeutically active polypeptide: An agent, such as an epitope of Mtb 25, that causes induction of an immune response, as measured by clinical response (for example increase in a population of immune.cells, increased cytolytic activity against Mtb, or=measurable reduction of a symptom of an infection). .
Therapeutically active molecules=can also be made from nucleic acids. Examples of a nucleic acid liased therapeutically active molecule is a nucleic acid sequence that encodes an Mtb epitope, wherein the nucleic acid sequence is operably linked to a control element such as a promoter. = = .

In one eiinbodiment, a therapeutically effective amount bfan Mtb polypeptide is an ainount used 'to generate ati 'immune response. =In several examples, "treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of tuberculosis.
5. ' = Therapeutically effective dose: A.dose sufficient to prevent advancement, or to cause regression of the disease, or which is capable of relieving-symptoms caused by - the disease: In one embodiment, a=therapeutically effective dose is a dose sufficient to prevent advancement or relieve symptoms of -tuberculosis.
Transduced and Transformed: A virus or vector "transduces' a cell when it transfers nucleic acid into the cell. A cell is "transformed' by a riucleic- acid t'ransduced into the cell. when the DNA becornes stably replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episoinal replication. As used he'rein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, iincluding transfection with viral vectors, transforriiation with plasinid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. = , . "
Tuberculosis (TB): A disease that is generally caused by Mycobacterium tub'erculosis that usually infects the lungs. However, other "atypical"
mycobacteria such as 11i kansasii may produce a similar clinical and pathologic appearance of =
disease. = = =
Transmission of M. tuberculosis occurs by the airborne route in confined ' areas with poor ventilation. In more than 90% of cases, following infection with M
tuberculosis, the immune system prevents development of disease from M.
25= tuberculosis, often called, active tuberculosis. However, not all of the M.
tuberculosis is-killed, and thus tiny, hard capsules are formed. -"Primary =.
tuberculosis" is seen disease that develops following an initial infection, usually.in children. The initial focus of infection is a small subpleural granuloma accompanied by granulomatous hilar lymph node infection.=Togetlier., these make up the Ghon 30. 'complex. In nearly all cases, these granulomas resolve and there is no further spread of the infection. "Secondary tuberculosis" is seen mostly in adults as a reactivation of previous infection (or reinfection), particularly when health status declines. The granulomatoius inflammation is much more florid and widespread. Typically, the upper lung lobes are most affected, and cavitation-can occur. Dissemination'of tuberculosis outside of lungs' can lead to the appearance of a nuirriber of uncommon findings'with characteristic patterns that include'skeletal tuberculosis, genital tract :tuberculosis,'urinary 'tract tuberculosis, central nervous system .(CNS) tuberculosis, gastrointestinal tuberculosis, adrenal tuberculosis, scrofula, and cardiac tuberculosis.
"Latent" tuberculosis is an Mtb infection in an individual that can be detected by a diagnostic assay, such- as, but not limited to a tuberculin skin test ~(TST) whetein the ' infection does not produce symptoms in that individual. "Active" hiberculosis is a symptomatia Mtb' infection in a subject.. = , Microscopically, the inflammation produced with TB infection is granulomatous, with epithelioid macrophages and Larighans giant cells along with lymphocytes, plasma cells, maybe a few polymorphonuclear cells, fibroblasts with collagen, and characteristic caseous necrosis in the center. The inflammatory response is mediated by a type IV hypersensitivity reaction, and skin testing is based on this reaction. -In some examples, tuberculosis can be d'iagnosed by a skin test, an acid fast. stain, an auramine stain, or a combination thereof. The-most common specimen screened is sputum, but the histologic stains can also be performed on tissues or other body fluids. . . =
. TB is a frequent.complication of HN infection. TB infection.in subjects infected with a human immunodeficiency virus (HIV) can spread readily and progress rapidly to active disease. Specif c* symptoms of lung disease due to Mtb infection include chronic cough and spitting blood. Other symptoms of TB
disease include fatigue, loss of appetite, weight loss, fever and drenching night sweats.
Vector: A nucleic acid molecule as intrdduced into a host cell, thereby =.producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
A.vector may also include one or more selectable marker gene and=other genetic elements known in the art. Vectors include plasmid vectois, including plasmids for expressioin in gram negative and gram positive bacterial cell. Exemplary Vectors include those for expression in E. coli and Salmonella: Vectors also include viral vectors, such as, but are not limited to, retrovirus, orthopox, avipox, fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus, baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors. Vectors also include vectors for expression in yeast cells Unless otlierwise explained, all technical and scientific terms used herein have the same meaning as commonly, understood by one of ordinary skill in the art to which this disclosure belongs. The singular=terms "a,' "an," and "the"
include..
plural referents. unless context.clearly indicates otherwise. Similarly, the word "or"
is intended to include ."arid" unless the context clearly indicatesotherwise.
It is fu.rther to be understood that all base =sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or-equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.
The=term "comprises" means "includes." All publications, patent applications, patents, and other references= mentioned herein are incorporated by reference in their entirety. In case of conflict; the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not iritende.d to be limiting.

Mycobacterium Polypeptides = .
- It is -disclosed herein that several Mycobacterium polypeptides can be used to induce an immune response to Mtb, such as a T cell response. The Mycobacterium polypeptides can be iused in diagnostic assays..to identify subjects infected with=a Ivlycobacterium such as Mtb. In several embodiinents, the polypeptide comprises or consists of the amino acid sequence set forth as:, .25= =

1. MXISRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISG

EQQEQASQQILS, (SEQ ID NO: 1, wherein Xl is A or T, X2 is T or A. and X3 is any amino acid, such as Q or no amino acid) ' ' In several exainples, the polypeptide'comprises or consists of the amino acid sequence set forth as: . ' a. MASRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGA
= ' GWSGMAEATSLDTMTQMNQAFRNIVNMLHGVRDGLVRDANNYEQ
QEQASQQILS (SEQ ID NO: 2) (See also= TUBERCULIST No. Rv1038c, as available ori March l; 2007, incorporated herein by reference, known as EsxJ, ES6 2, TB11. 0, 'QILSS) b. MASRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISG
AGWSGMAEATSLDTMAQMNQAFRNIVNMLHGVRDGLVRDANNYE
QQEQASQQILSS (SEQ ID NO: 3, TUBERCULIST No. *Rv1197, as available on March 1, 2007, incorporated herein by reference, also knovv as EsxK, ES6 3, TB11.0, QILSS) : =
c.. MASRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISG =
AGWSGMAEATSLDTMT+MNQAFRNIVNMLHGVRDGLVRDANNYE . =
QQEQASQQILSS (SEQ ID NO: 4, TUBERCLJLIST No. Rv 1992, as available on March i, 2007, incorporated herein by reference, as known as EsxM, TB11.0, QILSS. a. MATRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISG

AG W S GMAEATSLDTMAQMNQAFRNIVNMLHGVRDGLV RDANNYE
QQEQASQQILSS (SEQ ID NO: 5, TUBERCULIST No. Rv 234=7c, as == available on March 1, 2007, incorporated herein by reference, also known as EsxP, ES6 7, QILSS) . ' =

e. MTSRFMTDPHAMRDMAGRFEVHAQTVEDEARR1vIWASAQNISG
AGWSGMAEATSLDTMTQMNQAFRNIVNMLHGVRDGLVRDANNYE
. QQEQASQQILSS (SEQ ID NO: 6, TUBERCULIST No. Rv3620c, as _"
available on March 1, 2007, incorporated-herein by reference, also known as EsxW, ES6 10; QILSS). . = . = = .

Iri additional embodiments; the polypeptide comprises or consists of the,amino acid sequence set foith as:

2. IvISYMIATPAALTAAATDIDGIGSAVSVANAAAVAATTGVLAAGG
DEVLAAIARLFNANAEEYHALSAQVAAFQTLFVRTLTGGCGVFRRR
RGRQCVTAAEHRAAGAGRRQRRRRSGDGQW'=
RLRQQRHFGCGGQPEFRQHSEHRR (SEQ ID NO: 7, TUBERCULIST
NO. Rv1088, as available on March 1; 2007, incorporated herein by reference, also known as PE9). . :

3. VSLVIATPQLLATAALDLASIGSQVSA=ANA.AAAMPTTEVVAAAA
-DEV SAAIAGLFGAHARQYQALS V Q VAAFHEQFV QALTAAA.GRYAST
EAAV ERSLLGAVNAPTEALLGRPLIGNGADGTAPGQPGAAGGLLFG
= . . = NGGNGAAGGFGQTGGSGGAAGLIGNGGNGGAGGTGAAGGAGGNG
GW LWGNGGNGGV GGTS VAAGIGGAGGNGGNAGLFGHGGAGGTG
= . GAGLAGANGVNPTPGPAASTGDSPADVSGIGDQTGGDGGTGGHGTA
GTPTGGTGGDGATATAGSGKATGGAGGDGGTAAAGGGGGNGGDG
GVAQGDIASAFGGDGGNGSDGVAAGSGGGSGGAGGGAFVHIATAT
STGGSGGFGGNGAASAASGADGGAGGAGGNGGAGGLLFGDGGNG' GAGGAGGIGGDGATGGPGGSGGNAGIARFDSPDPEAEPDV VGGKGG
DGGKGGSGLGVGGAGGTGGAGGNGGAGGLLFGNGGNGGNAGAGG
DGGAGVAGGVGGNGGGGGTATFHEDPVAGVWAVGGVGGDGGSG
GSSLGVGGVGGAGGVGGKGGASGMLIGNGGNGGSGGVGCiAGGVG =
GAGGDGGNGGSGGNASTFGDENSIGGAGGTGGNGGNGANGGNGG =
AGGIAGGAGGSGGFLSGAAGVSGADGIGGAGGAGGAG.
GAGGSGGEAGAGGLTNGPGSPGVSGTEGMAGAPG (SEQ ID NO: 8, TUBERCULIST NO. Rv2487, as available on March 1, 2007, incorporated herein by reference, also known as PE PGRS42) =.

4. "MHQVDPNLTRRKGRLAALAIAAMASASLVTVAVPAI'ANADPEPA
PPVPTTAASPPSTAAAPPAPATPVAPPPPAAANTPNAQPGDPINAAPPP
ADPNA.PPPP.VIAP~TAPQPVRIDNPVGGFSFALPAGW VESDAABFDYG
SALLSKTTGDPPFPGQPPPVANDTRIVLGRLDQKLLYASAEATDSKAA
ARLGSDMGEFYMPYPGTRINQE`=TVSLDANGVSGSASYYEVKFSDPSK .
PNGQIWTGV IGSPAANAPDAGPPQRWFV V WLGTANNPVDKGAAKA
LAESIRPLVAPPPAPAPAPAEP APAPAPAGEVAPTPTTPTPQRTLPA .
(SEQ.ID NO: 9, TUBERCULIST No. Rv1860, as=available on March 1, 2007, incorporated herein by reference, .also known as Apa, modD; mpt32) 1.0 = ==
5. MLLALLRQHIRPYRRLVAMLMMLQLVSTLASLYLPTVIVAAIVDD
GVAKGDTATIVRLGAVMLGVTGLQVLCAIGAVYLGSRTGAGFGRDL
RSAMFEHIITFSERETARFGAPTLLTRSTNDVRQILFLVQMTATVLVT
APIMCVGGIIMAIHQEAALTWLLLVSVPILAVANYWIISHMLPLFRRM =
. 15. QSLIDGINRVMRDQLSGVRVVRAFTREGYERDKFAQANTALSNAAL
SAGNWQALMLPVTTLTINAS S VALIWFGGLRIDSGQMQVGSLIAFLS
YFAQILMAVLMATMTLAVLPRASVCAERITEVLSTPAALGNPDNPKF
PTDGVTGV VRLAGATFTYPGADCPVLQDISLTARPGTTTAIV GSTGS
'GKSTLVSLICRLYDVTAGAVLVDGIDVREYIHTERLWSAIGLV PQRSY
20 LFSGTVADNLRYGGGPDQVVTEQEMWEAL=RVA.AAD=GFVQTDGLQT
RVAQGGVNFS GGQRQRLAIARAV IRRPAIYVFDDAFSALDV HTDAK
VHASLRQV SGDATIIV VTQRISNAAQADQVIV VDNGKIVGTGTHETL
LADCPTYAEFAASQSLSATVGGVG (SEQ ID NO: 10, TUBERCULIST
NO. Rv 1273c, as available March 1, 2007, incorporated herein by 'reference).
25 =
6. MSYVIAAPEMLATTAADVDGIGSAIRAASASAAGPTTGLLAAAA
DEV S SAAAALFSEYARECQEV LKQAAAFHGEFTRALAAAGAAYAQ
AEASNTAAMSGTAGSSGALGSVGMLSGNPLTALMMGGTGEPILSDR
VLAIIDSAYIRPIFGPNNPVAQYTPEQW WPFIGNLSLDQSIAQGVTLLN

AFTLIGNINNPNGGVLERYVGLYLPFLDMSFNGATPPDSPYQTYMYT' GQYD GYAHNPQYPLNILSDLNAFMGIRW VHNAYPFTAAEVANAVPL

PTSPGYTGNTHYYMFLTQDLPLLQPIRAIPFVGTPIAELIQPDLRVLVD .
LGYGYGYADVPTPASLFAPINPIAVASALATGTVQGPQAALV SIGLLP
QSALPNTYPYLPSANPGLMFNFGQSSVTELSVLSGALGS VARLIPPIA
(SEQ ID NO: 11, TUBERCULIST NO. Rv01=59c, as available March 1, .5 = 2007, incorporated herein.by reference, also know as PE3 or PE): =

7. MEFPVLPPEINSVLMYSGAGSSPLLAAAAAWDGLAEELGSAAVSF
GQVTSGLTAGVWQGAAAAAMAAAAAPYAGWLGSVAAAAEAVAG ' QARV
VVGVFEAALAATVDPALVAANRARLVALAVSNLLGQNTPAIA

AAEAEYELMWAADVAAIVIAGYHSGASAAAAALPAFSPPAQALGGG .
VGAFLTALFASPAKALSLNAGLGNVGNYNVGLGNVGVFNLGAGNV
GGQNLGFGNAGGTNVGFGNLGNGNVGFGNSGLGAGLAGLGNIOLG
NAGS SNYGFANLGVGNIGFGNTGTNNVGVGLTGNHLTGIGGLNSGT
GNIGLFN S GTGN V GFFNS GTGNFGV FNS GNYNTGV GNAGTASTGLF
NA:GNFNTGV VNV GSYNTGSFNAGDTNTGGFNPGGVNTGWLNTGNT
NTGIANSGNVNTGAFIS GNFNNGV LW V GDYQGLFGV SAGS SIPAIPIG
LVLNGDIGPITIQPIPILPTIPLSIHQTVNLGPLV VPDIVIPAFGGGIGIPIN
=IGPLTITPITLFAQQTFVNQLPFPTFSLGKITIPQIQTFDSNGQLVSFIGPI
VIDTTIPGPTNPQIDLTIRWDTPPITLFPNGI SAPDNPLGLLV S V SISNPG
= .. FTIPGFSVPAQPLPLSIDIEGQIDGFSTPPITIDRIP.LTVGGGVTIGPITIQG
L HIPAAP GV GNTTTAP S S GFFN S GAGGV S GFGN V GAG S S G W W NQAP
SALLGAGS.GV GNVGTLGSGVLNLGSGISGFYNTS VLPFGTPAAVSGI
GNLGQ QL S GV SAAGTTLRS MLAGNLGLAN V GNFNT.GFGN V GD VNL.
GAANIGGHNLGLGNV GDGNLGLGNIGHGNLGFANLGLTAGAAGV G
. '. NVGFGNAGINNYGLANMGVGNIGFANTGTGNIGIGLVGDHRTGICiG
LNSGIGNIGLFNSGTGNVGFFNSGTGNFGIGNSGRFNTGIGNSGTAST
GLFNAGSFSTGIANTGDYNTGSFNAGDTNTGGFNPGGINTGWFNTGH
ANTGLANAGTFGTGAFMTGDYSNGLLWRGGYEGL`TGVRV GPTIS QF
PVTVHAIGGV GPLHVAPVPVPAVHVEITDATV GLGPFTVPPISIPSLP
IASITGSVDLAANTISPIRALDPLAGSIGLFLEPFRLSDPFITIDAFQV VA
GV LFLENIIVPGLTV SGQILVTPTPIPLTLNLDTTPWTLFPNGFTIPAQT

PVTVGMEVANDGFTFFPGGLTFPRASAGV TGLSV GLDAFTLLPDGFT-LDTVPATFDGTILIGDIPIPIIDVPAVPGFGNTTTAPS SGFFNTGGGGGS
GFANV GAGTSGW WNQGHDVLAGAGSGVANAGTLSSGVLNVGS
GISGWYNTSTLGAGTPAV V S GIGNLGQQLS GFLANGTV LNRSPIVNIG
WADVGAFNTGLGNVGDLNWGAANIGAQNLGLGNLGSGNVGFGNIG =
AGNVGFANSGPAVGLAGLGNVGLSNAGSNNWGLANLGVGNIGLAN
TGTGNIGIGLV GDYQTGIGGLNS GSGNIGLFNS GTGNV GFFNTGTGNF-GLFNSGSFNTGGNSGGTGSTGLFNAGNFNTGIANPGSYNTGSFNVGDT
NTGGFNPGDINTGWFNTGIMNTGTRNTGALMSGTDSNGMLWRGDH
EGLFGLSYGITIPQFPIRITTTGGIGPIVIPDTTILPPLHLQITGDADYSFT=
VPDIPIPAIHIGINGV VTVGFTAPBATLLSALKNNGSFISFGPITLSNIDIP
PIVSDFTLGLPVLGPITGQLGPIHLEPIVVAGIGVPLEIEPIPLDAISLSESIP , IRIPVDIPAS V IDGISM SEV VPIDAS VDIPAVTITGTTISAIPLGFDIRTSA
GPLNIPIIDIPAAPGFGNSTQMPSSGFFNTGAGGGSGIGNLGAGV SGLL
NQAGAGSLVGTLSGLGNAGTI.ASGVLNSGTAISGLFNVSTLDATTPA
VISGFSNLGDHMSGV SIDGLIAILTFPPAESVFDQIIDAAIAELQHLDIG
NALALGNV GGVNLGLANV GEFNLGAGNV GNINV GAGNLGGSNLGL
GNVGTGNLGFGNIGAGNFGFGNAGLTAGAGGLGNYGLGNAGS
GS WGLANV GVGNIGLANTGTGNIGIGLTGDYRTGIGGLNSGTGNLGL
, FNSGTGNIGFFNTGTGNFGLFNSGSYSTGVGNAGTASTGLFNAGNFN =
= TGLANAGS YNTGSLNV GSFNTGGVNPGTVNTGWFNTGHTNTGLFNT
GNVNTGAFNSGSFNNGALWTGDYHGLV GFSFSIDIAGSTI.LDLNETL
NLGPII:iIEQIDIPGMSLFDVHEIVEIGPFTIPQVDVPAIPLEIHESIHMDPI
- VLVPATTIPAQTRTIPLDIPASPGSTMTLPLISMRFEGEDWILGSTAAIP
NFGDPFPAPTQGITIHTGPGPGTTGELKISIPGFEIPQIATTRFLLDVNIS
GGLPAFTLFAGGLTIPTNAIPLTIDASGALDPITIFPGGYTIDPLPLHLAL
NLTVPDSSIPIIDVPPTPGFGNTTATPSSGFFNSGAGGVSGFGNVGSNL= '=
SGWWNQAASALAGSGSGVLNVGTLGSGVLNVGSGVSGIYN = .
TS V LPLGTPAVLSGLGNV GHQLSGV SAAGTALNQIPILNIGLAD V GNF
.30 NVGFGNVGDVNLGAANLGAQNL'GLGNVGTGNLGFANVGHGNIGFG
NS GI.,TAGAAGLGNTGFGNAGSANYGFANQ GVRNIGLANTGTGNIGI
= GLVGDNLTGIGGLNSGAGNIGLFNSGTGNIGFFNSGTGNFGIGNSGSF.

-34- =

NTGIGNS GTGSTGLFNAGSFNTGVANAGSYNTGSFNAGDTNTGGFNP
GTINTGWFNTGHTNTGIANS GNVCiTGAFMS GVFSNGLL WRGDHEGL
FSLFYSLDVPRITIVDAHLDGGFGPV V LPPIPVPAVNAHLTGNVAMGA

5' DPFEMWTQGTNGLGITFYSFGSADG'rSPYATGPLVFGAGTSD : ' . ' GSHLTISASSGAFTTPQLETGPITLGFQVPGSVNAITLFPGGLTFPATSL
LNLDVTAGAGGVDIPAIT:WPEIAASADGS VYVLASSIPLINIPPTPGIG
NSTITPS SGFFNAGAGGGSGFGNFGAGTSGW WNQAHTALAGAGS GF
' ANVGTLHSGVLNLGSGVSGIYNTSTLGVGTPALVSGLGNVGHQLSG
:10 . . LLSGGSAVNPVTVLNIGLANVGSHNAGFGNVGEVNLGAANLGAHNL
GFGNIGAGNLGFGNIGHGNVGVGNSGLTAGVPGLGNVGLGNAGGN
NWGLANVGVGNIGLANTGTGNIGIGLTGDYQTGIGGLNSGAGNLGL
FNSGAGNVGFFNTGTGNFGLFNSGSFNTGVGNSGTGSTGLFNAGSFN
TGVANAGSYNTGSFNV GDTNTGGFNPGS INTGWLNAGNANTGVAN

VSGGIGPITVLPPIHIPPIPVGFAAVGGIGPIAIPDISVPSIHLGLDPAVHV .
= GSITVNPITVRTPPVLVSYSQGAVTSTSGPTSEIWVKPSFFPGIRIAPSS
GGGATSTQGAYFVGPISIPSGTVTFPGFTIPLDPIDIGLPVSLTIPGFTIP
GGTLIPTLPLGLALSNGIPPVDIPAIVLDRILLDLHADTTIGPINVPIAGF

-GSASGFANFGTQLSGILNRGAGISGVYNTGALG=V VTAAVVSGFGNV
GQQLSGLLFTGVGP (SEQ ID NO: 12, TUBERCULIST'No. 3350c, as ' available March 1, 2007, herein incorporated by reference, also known as PPE56 or PPE.
25 . = In a second embodiment,'an Mtb polypeptide of use in=the methods disclosed herein has a sequence at least 75%, 85%, 90 do; 95%, 96%, 97%, 98% or 99%
homologous =to the amino acid sequence set forth in one of SEQ ID NOs: 1-12.
For example; the.polypeptide can have an amino* adid sequence, at least 85%, 90 fo,'95%, 96%, 97%, 98% or 99 1o homologous to one of the amino acid sequerices set forth in 30 ' SEQ ID NOs: 1-12. Exernplary sequences can be obtained using computer progfams that are readily available on the internet and the amino acid sequences set forth herein. In one example, the polypeptide retains a function of the Mtb protein, such as binding to an antibody that specificallybinds the.lVltb.epitope. .
Mirior modifications of an Mtb polypeptide primary amino acid sequences rriay result in peptides whicli have substantially equivalent activity as compared to =
5' =the unmodified counterpart polypeptide. described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein. Thus, a specific, non-limiting example of an Mtb polypeptide is a conservative variant of the Mtb polypeptide. A table of conservative substitutions is provided herein.
Substitutions=
of the amino acids sequence shown in SEQ ID NOs: 1-12 can be made based on this table.
Mtb polypeptides are'disclosed herein that can be used to detect an immune response to Mtb. These peptides include or consist of at least nine amino acids, such as nine to twenty amino acids consecutive amino acids of an Mtb polypeptide set forth above. Specific, non-limiting examples are twelve, eleven, ten amino acids, or nine consecutive amino acids of one of the Mtb polypeptides set 'forth above.
In these examples, the Mtb polypeptide does not include the full-length amino acid =sequences set forth as SEQ ID NOs: 1-1 2. =
An isolated polypeptide is disclosed that includes nine to twelve consecutive arnino acids from an Mtb. -polypeptide, wherein the isolated'.polypeptide comprises the amino acid sequence set forth as QTVEDEARRMW (SEQ ID NO: 13). In some embodiments, the polypeptide is nine, ten or eleven amino acids in length. In additional embodiments,.the polypeptide consists of the amino acid sequence set forth as SEQ ID NO: 13. An isolated polypeptide is disclosed that includes nine to 25, twelve consecutive amino acids from an Mtb polypeptide, wherein -the isolated polypeptide comprises the amino acid sequence set forth as VSAAIAGLF (SEQ ID
NO: 14). In some.embodiments, the polypeptide is nine, ten or eleven amino acids in length. In additional embodiments, the polypeptide consists of the amino acid sequence set forth as SEQ ID NO: 14. .
.. In several embodiments, the isolated Mtb polypeptide is included in a fusion protein. Thus, the fusion protein can include the Mtb polypeptide (see above) and a second heterologous moiety, such as a myc protein, an enzyme or a carrier (such as a hepatitis carrier protein or bovine serum albumin) covalently linked to the.
Mtb-polypeptide.' In several examples, a polypeptide consisting of=nine to twelve amino acids of one of the amino acid sequences set forth as SEQ ID NOs: 1-14 that bind. =.
MHC=class I is covalently linked to a carrier. In additional example, a polypeptide =
:5 consisting of one of the amino acid sequences set forth as one of SEQ ID
NOs: 1-14 is covalently linked to a carrier.
In additional exarnples, the polypeptide ca.in be, a fusion protein and can also include heterologous sequences to Mtb=(such as amino acid sequences of at least nine: amino acids in length that are not included in SEQ ID NO: 1). Thus, in several =
specific non-limiting.'examples, the immunogenic peptide is a fusion polypeptide, for example :the polypeptide includes six sequential histidine residues, a(3-galactosidase amino acid sequence, or an immunoglobulin amino acid sequence.
The polypeptide can also be covalently linked to a carrier. In additional .
embodiments, the protein consists of the Mtb polypeptide.
The polypeptide can optionally include repetitions of one or more of the Mtb poiypeptides disclosed herein. In one specific, non-limiting example, the polypeptide includes two, three, four, five, or iip to ten repetitions of one'of the Mtb polypeptides described above. = Alternatively, more thanone polypeptide can be included in a fusion polypeptide. Thus, in several examples, the polypeptide can include at least two, at least 'three, at least four, at least =five or at least six of the amino acid sequences set forth as SEQ ID NOs: 1-14. A.linker sequence can optionally be included between the Mtb polypeptides. The Mtb polypeptides disclosed herein can be chemica.lly synthesized by standard methods, or can be produced recombinantly. An exemplary process for . polypeptide production is described in Lu et al., Federation of European Biochemical Societies Letters. 429:31-35, 1998. They can also be isolated by =
methods including preparative chromatography and immunological separations.
If desired, polypeptides can also be chemically synthesized by ernerging techriologies. One such process is described iri W. Lu et id., Federation of European Biochemical Societies Letters. 429:31-35, 1998. Polypeptides can also be produced using molecular genetic techniques, such as by inserting a nucleic acid encoding'Mtb or an epitope thereof -into an expressioin .vector; introducing the .'.
expression vector into =a host 'cell, and isolating the polypeptide (see=
below).
.'. Polynucleotides encoding the Mtb polypeptides disclosed herein*are also provided. Exemplary nucleic acid sequences are set forth below:
ESXJ=(ESAT-6 LIKE PROTEIN 2)=
atggcctcgcgttttatgacggatccgcacgcgatgcgggacatggcgggccgttttgag gtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatc tcgggcgcgggctggagtggcatggccgaggcgacctcgctagacaccat'gacccagatg aatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgc gacgccaacaactacgaacagcaagagcaggcctcccagcagatcctcagcagctga=
(SEQ ID NO: 15) ESXK (ESAT-6 LIKE=PROTEIN=3) =
.atggcc.tcacgttttatgacggatccgcacgcgatgcgggacatggcgggccgttttgag gtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcggcaaaacatt tccggtgcgggctggagtggcatggccgaggcgacctcgctagacaccatggcccagatg aatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgc gacgccaacaactacgagcagcaagagcaggcctcccagcagatcctcagcagctaa (SEQ ID NO: 16) =

ESXM (ESAT-6 LIKE PROTEIN ESXM).
atggcctcacgttttatgacggatccgcatgcgatgcgggacatggcgggccgttttgag gtgc=acgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatt tccggtgcgggctggagtggcatggccgaggcgacctcgctagacaccatgacctagatg aatcaggcg'tttcgcaacatcgtgaacatgctgcacgg.ggtgcgtgacgggctggttcgc gacgccaacaactacgaacagcaagagcaggcctcccagcagatcctgagcagctag (SEQ ID NO: 17) ESXP (ESAT-6 LIKE PROTEIN 7) atggcaacacgttttatgacggatccgcacgcgatgcgggacatggcgggccgttttgag gtgcacgccicagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatc tcgggcgcgggctggagtggcatggccgaggcgacctcgctagacacca=tggcccagatg=
=aatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgc gacgccaacaactacgagcagcaagagcaggcctcccagcagatcctcagcagctaa (SEQ ID NO : 18) ' . =

ESXW (ESAT-6 LIKE PROTEIN 10) atgacc.tcgcgttttatgacggatccgcacgcgatgcgggacatggcgggccgt=tttgag gtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatt.
tccggcgcgggctggagtggcatggccgaggcgacctcgctagacaccatgacccagatg aatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgc.
gacgccaacaactacgaacagcaagagcaggcctcccagcagatcctcagcagctga (SEQ ID NO: 19) =

PE9 (PE FAMILY PROTEIN) =
atgtcatacatgattgccacaccagcggcgttgacggcgggcggcaacggat=atcgacggg attggctcggcggttagcgttgcgaacgccgcggcggtcgccgcgacaaccggagtgctg gccgccggtggcgatgaagtgttggcggccatcgctaggctgttcaacgcaaacgccgag gaatatcacgccctcagcgcgcaggtggcggcgtttcaaaccctgtttgtgcgcaccttg actggggggtgcggagtctttcgccggcgccgaggccgccaatgcgtcacagctgcagag =catcgcgcggcaggtgcggggcgccgtcaacgccgtcgccggtcaggtgacgggcaatgg cggctccggcaacagcggcacttcggctgcggcggccaacccgaattccgacaacacagc Gagcatcgccgatag (SEQ ID NO: 20) PE_PGRS42 (PE-PGRS FAMILY PROTEIN) gtgtcgttggtgatcgcgacgccgcagctgctggcaactgcggctttggatttagcgagt =attggttcgcaggtgagcgcggctaatgcggccgcggcgatgccgacgacggaagtggtg gctgcggctgccgatgaagtgtc=ggcggcgattgcggggttgttcggggcccatgctcgg =
ca.gtatcaggcgctcagcgtacaggtggcagcgtttcacgagcagtttgtgcaggcgt"tg actgcggccgcgggtcggtat=gccagcactgaggccgctgttgagcggagtctgctgggt gcggtgaatgcgcecaccgaggcgcttttggggcgcccgttgatcggaaacggcgccgac gggacggcacccgggcagcctggcgcggccggcgggttgctgtttggc=aacggtggcaac ggcgcggctggcgggttcggtcaaaccggcggcagcggaggcgcggccgggttgatcggc aacggcggcaacggcggggccggtggtaccggcgcggccggcggtgccggtgggaacggg gggtggttgtggggcaacggcggcaacggcggtgtcggcggcaccagcgtggccgcaggc=.
atcgggggtgcgggcggtaacggcggcaacgccgggctgttcggccatggcggcgccggt.
.ggtaccggcggcgccggcctcgccggggcaaacggggtcaatcccacgcccggccccgcg=
=gccagcaccggggacagcccggcagatgtgtccggcatcggtgatcaaaccggcggcgac .ggcggcacgggcggccatggcactgccggcacgccgaccggtggcaccggcggcgacggt gccaccgcgacggcaggctcgggcaaggccaccggcggtgccggtggtgacggcggtacc ..gccgctgccggtggcggcggcggcaacggcggcgacggcggagtcgcgcagggcgacatt gcgagcgcctttggcggtgatggtggcaacgggtccgacggtgtagccgccggcagtggg ggtggtagcggcggcgccggaggcggcgctttcgtacacatcgccactgccacctctacc ggtggtagcggcggtttcggtggtaacggggctgccagtgccgcctccggcgccgacggt ggcgcagggggagctggcggcaatggtggcgccggcgggttgctattcggtgatggcggc aacggtggcgccggtggcgcgggtggtatcggtggtgacggcgccac'gggggggcccggg ggaagcggcggcaacgctggcatcgcgaggtttgacagcccagaccccgaggcagaaccc gatgtggtcggcggcaagggtggtgatggcggcaagggcggcagcggccttggcgtcggc ggcgccggcgggaccggcggcgcgggcggcaacggcggcgccggcgggttgttgttcggc.=
aacggcggcaacggcggcaacgccggggccggcggggatggcggcgccggcgttgccggt gyggttggcggtaacggcggcggtggtggcaccgcgacgtttcacgaagacccgg.tcgct ggtgtctgggcggtcggtggcgtaggtggtgatggtggctccggcggcagctcgcttggt gtcggcggggtgggcggagccggtggcgtgggtgg=caagggtggcgccagcggcatgttg atcggcaacggcggcaacggtggcagcggcggagtcggtggggccggtggagtcggcggg gctggcggtgacggcg.gcaacggcggctccggtggcaacgccagtacttttggcgatgag.
aactccatcggciggggccggcgggacgggcggcaacgggggcaacggcgcaaacggcggt aacggtggcgctggcggtattgccggcggtgcgggtgggtccggagggttcctcagcggt gccgcaggagtcagcggcgctgacggtatcggtggcgcgggcggcgcaggcggtgccggt ggcgcgggcggtagcggcggtgaggcaggcgcggggggcctcaccaacggccccgggtcc cctggcgtttccggcaccgaaggcatggccggcgcgcccggctag (SEQ ID NO: 21) Rv1860 '(FIBRONECT IN ATTACHMENT PROTEIN) atgcatcaggtggaccccaacttgacacgtcgcaagggacgattggcggcactggctatc.
gcggcgatggccagcgccagcctggtgaccgttgcggtgcccgcgaccgccaacgccgat ccggagccagcgcccccggtacccacaacggccgcctcgccgccgtcgaccgctgcagcg ccacccgcaccggcgacacctgttgcccccccaccaccggccgccgccaacacgccgaat gcccag.ccgggcgatcccaacgcagcacctccgccggccgacccgaacgcaccgccgcca cctgtcattgccccaaacgcaccccaacctgtccggatcgacaacccggttggaggattc agcttcgcgctgcctgctggctgggtggagtctgacgccgcccacttcgactacggttca gcactcctcagcaaaaccaccggggacccgccatttcccggacagccgccgc=cgg=tggcc aatgacacccgtatcgtgctcggccggctagaccaaaagctttacgccagcgccgaagcc accgactccaaggccgcggcccggttgggctcggacatgggtgagttctatatgccctac .ccgggcacccggatcaaccaggaaaccgtctcgctcgacgccaacggggtgtctggaagc' =gcgtcgtattacgaagtcaagttcagcgatccgagtaagccga'acggccagatctggacg ggcgtaatcggctcgcccgcggcgaacgcaccggacgccgggccccctcagcgctggttt*
gtggtatggctcgggaccgccaacaacccggtggacaagggcgcggccaaggcgctggcc gaatcgatccggcctttggtcgccccgccgccggcgccggcaccggctcctgcagagccc gctccggcgccggcgccggccggggaagtcgctcctaccccgacgacaccgacaccgcag Cggacc,ttaccgg'cctga , (SEQ.=ID NO:= 22) =

Rv1273c.(PROBABLE DRUGS-TRANSPORT=TRANSMEMBRANE ATP-BINDING.=, PROTEIN ABC TRANSPORTER) " =
atgctcctggccctgctgcgccagcacatccgaccgtaccgccggctggtcgcgatgctg atgatgctgcagctggtcagcaccctggct=tcgctatacctcccgacggtcaacgccgca =
atcgtcgacgacggcgtcgc.caagggcgacaccgccaccatcgtacggctgggtgcggtg atgcttggggtga.ccggattgcaggtgctgtgcgcgatcggggcggtctatctgggctcc cggaccggggcgggtttcggccgtgacctgcgctcggcaatgttcgaacacatcatcacc ttctcggaacgcgagaccgcccgattcggcgctccgacgttgttgacccggcagcaccaac gacgtccggcagatcctgttcctggtccagatgaccgccaccgtgctggtcaccgcaccg atcatgtgcgtcggcggaatcatcatggccatccaccaggaggccgcgctgacatggctg' ctgc=tggtcagcgttccga.ttctggccgtagcaaactactggatcatctcccacatgctg ccgctcttccgccgcatgcagagcctgatcgacggcatcaaccgggtgatgcgcgatcag =ctgtccggggtgcgagtggtccgcgccttcacccgcgaaggctatgaacgcgacaagttc gcgcaggccaatacggcgctgtcgaatgccgcactgagcgccggcaactggcaagcactg atgctgccggtgaccacgctgaccatcaacgcatccagcgtcgcactgatctggttcggt gggctacgcatcgacagcggccagatgcaggtcggctccctgatcgccttcctgtcctac ttcgcccagatcctgatggcggtgttgatggcgaccatgacgctggccgtgctgccacga gcgtcggtctgcgccgaacgcatcaccgaggtgctttccacgcccgccgcactcggtaac cccgacaatcccaagttcccgacggacggggtcacgggcgtagtgcgct.tggctggcgca acctttacctatcctggcgccgactgcccggtgctgcaggacatttcgttgactgcgcgg =cccggtaccaccaccgcgatcgtcggcagtaccggttcgggcaagtcgacactggtgtcg ttgatctgccggctctacgacgtcaccgctggcgcggtcttggttgacggtatcgacgtc cgcgagtaccacaccgagcggctctggtcagcgatcgggctggtgccccagcgcagctac .ctcttctccggaaccgtcgcggacaacctgcgctacggcgggggcccagaccaggtagtc accgagcaggagatgtgggaggcgctgcgggtcgccgcggccgacggctttgtacaaaca gacgggctgcagacgcgtgtcgcccaaggtggtgtcaacttctccggcgggcagcgccaa cggctggcgatagcccgagcggtcatccgacgtccggccatctatgtgttcgacgacgcg ttctccgcacttgacgtgcacaccgacgccaaagtccacgcatcgctgcgacaggtatat ggtgatgcaaccatcattgttgttacacaacggatttcgaatgccgctcaggccgaccag.=
gtcatcgttgtcgataacggtaagatcgtcggcacgggcacccacgaaacgctgctggcc gattgccccacctatgccgaattcgccgcctcacaatcgctgagcgccacggtcgggggt Gtagggtga (SEQ ID NO: 23) = . .
Rv0159c (PE FAMILY PROTEIN) . = =
atgtcctacgtcatcgcggccccggagatgttggcaacgacggccgcggacgtggacggg atcggttcggcgatacgagcggccagcgcgtccgctgcgggtccaacgacc=ggactgctg gccgcggccgccgatgaggtgtcgtcggccgctgcagcgctgttcagcgaatacgcgcgc, gaatgtcaag=aggtcctaaagcaggctgcggcgttccatggcgagttcacccgggcgctg gctgccgccggggccgcctatgcccaggctgaagccagcaacaccgctgctatgtcgggc accgccgggtccagcggcgccctcggttctgtcgggatgctgtcaggcaacccgctaacc gcgttgatgatgggcggcaccggggaaccgatccttagtgaccgcgtcttggcgatcatt gacagcgcatacattcggcccattttcgggcccaacaacccggtcgcccagtacacgccc gagcagtggtggccgtttatcgggaacctgtcactggaccaatccatcgcccagggtgtc acgctgctgaacaacggcatcaacgcggaactacaaaatgggcatgacgtcgtcgttttc ggctactcgcaaagcgccgcggtagcgaccaatgaaatacgcgctcttatggcgttacca ccgggccaagccccagatccaagccggctggctttcacgttgatcggtaatatcaataac cccaacggcggcgtcctcgagcgttacgtgggcctttacctcccgttcttggatatgtcg=
ttcaacggtgcgactccaccggattccccctaccagacctacatgtacaccggccaatac gacggctacgcccacaacccgcagtacccgctcaatatcttgtcggacctcaacgccttc atgggcatcagatgggtgcacaacgcgtaccccttcaccgcggccgaggttgccaatgcc gtgccgttgcccacgtctccgggctacaccggcaacacccattactacatgtttctgacc caggacctgccgctgt'tgcagccgattcgcgccatccccttcgtagggaccccaatagcc gagctgattcagcccgacctacgggtgctagtcgacttgggctatggctacggctacgcc gacgtacccaccccggccagcctgttcgcgccaatcaacccgatcgccgtggcctcggcc ctggcgaccgggacc=gtgcaaggcccccaagccgccctagtaagcatcggattg.ttaccg.
cagtccgcgctacccaatacgtatccgtatcttccgtcggcgaatccgggcctgatgttc;.
aacttcggtcaatccagtgtgacggagttgt.cggtgctcagtggcgccctcgggtccgta gcgagattgattccaccgatcgcgtga .
(SEQ ID= NO: 24);

Rv3350c (PPE FAMILY.PROTEIN)= = . = ' atggagtttccggtgttgccaccggaaatcaactccgtgctgatgtattcgggtgcgggg tcgagcccgttgctggcggcggccgcggcgtgggatgggctggctgaggagttggggtcg, gcggcggtgtcgtttgggcaggtgacgtcgggcctgacggcgggggtgtggcagggtgcg .gcggcggcggcgatggcggccgcggcggqgccgtatgcggggtggttgggttcggtggcg', =gccgcggccgaggcggtggccgggcaggcgcgggtggtggtgggggtctttgaggcggcg.
ttggcggcgacggtggatccggcgctggtggcggccaaccgggcgcggctggtggcgttg .gcggtgtcgaatctgttggggcagaacacgccggcgatcgcggccgccgag.gccgagta=c gagctgatgtgggccgccgatgtggcggcgatggccggctaccattccggcgcgtcggct gctgccgcggcgttgccggcgttcagcccaccggcgcaggcgctggggggaggtgtcggc gcgttccttaccgccctgttcgccagccctgcgaaggcgctgagcctgaatgcgggtttg ggcaatgtcggcaattacaacgtcgggttgggcaatgtcggggtgttcaacctgggcgcg ggcaatgtgggtgggcagaatctgggtttcgggaatgccggtggcaccaatgtcgggttc ggcaa.cctcggtaacgggaatgtcgggttcggcaactccggtc=tgggggcgggcctggcc, ggcttgggcaatatcgggttgggcaatgcgggcagcagcaactatggtttcgcaaacctg ggtgtgggcaacatcggtttcggcaacaccggcaccaacaacgtcggcgtcgggctcacc ggcaaccacctgacgggtatcgggggcctgaattcgggcaccgggaatatcgggttgttc aactccggcaccgggaatgtggggtt'cttcaattcggggaccgggaacttcggggtgttc aactcgggtaattacaacaccggtgtcggtaatgcggggacgg.ccagcacggggttgttc aatgccggcaatttcaacaccggcgtggtgaacgtgggeagttacaacaccggcagtttc aacgccggcgacaccaacaccggtggcttcaaccccggcggtgtgaacaccggctggctg aacaccggcaacaccaacaccggcatcgccaactcgggcaacgtcaacaccggcgcgttc atctcgggcaacttcaacaacggcgtgctgtgggtgggtgactaccagggcctgttcggc=
gtctccgcc.ggctcgtcgatccccgcaattcccatcggcctggtgctcaacggcgacatc ggcccgatcaccatccagcccatcccgatcctgcccaccatcccgc.tcagcattcaccaa accgtcaacttgggcccgctggtggttcccgacatcgtgatccccgccttcggcggcggt atcggcatacccatcaacatcggcccgctgaccatcacacccatcaccctgtttgcccaa cagacatttgtcaaccaattgccctttcccaccttcagtttagggaa.aatcacaattcca caaatccaaacctttgattctaacggtcagcttgtcagctttatcggccctatcgttatc .gacaccaccattcccggacccaccaatccacagattgatttaacgatcagatgggatacc=
cctccga=tcacgctgttcccgaatggcatcagtgctcccgataatcctttggggttgctg =
gtgagtgtgtcgatcagtaacccgggctttaccatcccgggatttagtgttcccgcgcag' ccgttgccgttgtcgatcgatatcgagggccagat'cgacgggttcagcaccccgccgatc .acgatcgatcgcatccccctgaccgtggggggcggggtcacgatcggccccatcacgatc cagggccttcatatcccggcggcgccgggagtggggaacaccaccacggccccgtcgtcg ggattcttcaactccggtgcgggtggggtgtcgggtttcggcaacgtcggcgcgggcagc tcgggctggtggaaccaggcgccgagcgcgctgttgggggccggttcgggtgttggcaac gtgggcaccctgggctcgggtgtgctcaacctgggctcagggatctcggggttctacaac accagcgtgttgcctttcgggacaccggcggcggtgtcgggcatcggcaacctgggccag cagctgtcgggggtgtcggcggcgggaaccacgctgcgctcgatgctcgccggcaacctc gggttggccaatgtgggcaacttcaacaccgggttcggaaatgtcggggacgtcaaectg ggtgcggccaacatcggtgggcacaacctgggcctgggcaatg.tcggggacggcaacctg'=
gggt=tgggcaacatcggccatggcaacct=gggggtttgccaacttgggcctgaccgccggc gcggcgggggtgggcaatgttggtt=ttggcaatgccggcatcaacaactatggcttggcg aacatgggtgtgggcaatattgggtt=tgccaacaccggcacgggcaacatcgggatcggg ctggtcggggaccatcggaccgggatcgggggcttgaactccggcatcggcaatatcggg ttgttcaactccggcaccggcaacgtcgggttcttcaattccgggaccggcaacttcggc=
atcgggaactccggccgcttcaacaccgggatcggtaatagcggaacggccagcaccggg ctcttcaatgccggcagcttcagcaccggcatcgccaacactggtgactacaacacgggc agcttcaacgccggcgacaccaacaccggtggcttcaacccgggcggcatcaacaccggc tggttcaacaccgggcatgccaacaccgggttggccaacgcgggcaccttcggcaccggc gccttcatgacgggcgactacagcaacggcctgttgtggcggggcggctacgagggcctg' gtcggcgtccgcgtcgggcccacgatctcccaattcccggtcaccgtgcacgc'gatcggc ggggtgggcccgctgcatgtggcgcccgtcccggtacccgccgtgcacgtcgagatcacc gacgccaccgtcggcctgggtccgttcaccgtcccaccgatcagcattccctcacttccc atcgccagcatcaccggaagcgtggacctggccgcaaacaccatctcgccgattcgcgct cttgacccgctcgccggttcgatagggctttttctcgagccgttccgcctcagtgaccca' ttt-atcaccattgatgcgttccaagttgttgccggtgtc.ttgt=tcctagagaacatcatt=
gtgcccggcctcacggttagcggtcagatattggtcaccccgacaccaattcccctaacc ctcaacttggacaccaccccgtggacjgcttttcccgaatggtttcaccattcccgcgcaa acccccgtgacggtgggtatggaggtcgccaacgacgggttcaccttcttcccgggtggg ctgacctttccgcgggcctccgccggggtcaccggactgtccgtggggctggacg`cgttc acgctgttgcccgacgggttcaccctcgacac'cgtgccggcgaccttcgacggcaccatc ctcatcggcgatatcccgatcccgatcatcgatgtgccggcggtgccggggttcggcaac accaccacggccccatcgtcggggttcttcaacaccggcggcggcggtggatcggggttc gccaacgtcggcgcggggcacgtcgggctggtggaaccaggggcacgacgtgttagcaggg gcgggctcgggagttgccaatgccggcacgctgagctcgggcgtgctgaacgtcggctcg gggatctccgggtggtacaacaccagcaccctgggagcgggcaccccggcggtggtctcg ggcatcggcaacctcggccagcagctgtcggggttcttggcaaatgggaccgtgctcaac cggagccccattgtcaatatcgggtgggccgatgtgggcgcgttcaacaccgggttgggc aatgtgggggacctcaactggggtgcggccaacatcggcgcgcagaacctgggcctgggc .aatctcggcagcgggaacgtcgggttcggcaacatcggtgccggcaacgtcgggttcgcc aactcgggtccggcggtgggcctggccggcctgggcaacgtggggttgagcaatgccggc agcaacaactgggggctggccaacctgggtgtgggcaacatcgggttggccaacaccggc acgg.gcaacatcgggatc'gggctggtcggcgactaccagaccggcatcggcggcctcaac tcgggtagtggcaatatcggattgttcaattccggcaccggcaatgtcgggttcttcaac accggcaccggcaacttcggactgttcaactccggtagtttcaacaccggcatcggtaat agcggaaccggcagtactgggctcttcaatgccggcaatttcaacaccggcatcgccaac cccgggtcgtacaacacgggcagcttcaatgtcggtgataccaacaccggtggtttcaac ccgggcgacatcaacaccggctggttcaacaccggcattatgaatacgggcacccgcaac accggcgccctcatgtcggggaccgacagcaacggcatgctgtggcgcggcgaccacgag ggcctgttcggcctgtcctatggcatcacgatcccgcaattcccgatccgcatcaccacg actggcggtatcggccccatcgtcatcccggacaccacgatccttccgccgctgcacctg==, cagatcaccggcgacgcggactacagcttcaccgtgcccgacatccccatccccgccatc cacatcggcatcaatggcgtcgtcaccgtcggcttcaccgccccggaagccaccctgctg tccgccctgaagaataacggtagcttcatcagcttcggccccatcacgctctcgaatatc=
gatattccgcccatggatttcacgttaggcctgcccgttcttggtcctatcacgggccaa ctcggaccaattcatcttgagccaatcgtgg=tggccgggatcggtgtgcccctggagatc gagcccatccccctggatgcgatttcgttgagtgagtcgattcctatccgcatacctgtt=
gatattccggcctcggtcatcgatgggatttcaatgtcggaagtggtgccgatcgatgcg tccgtggacatcccggcggtcacgatcacaggcaccaccatttccgcgatcccgctgggc 'ttcgacattcgcaccagtgccggacccctcaacatcccgatcatcgacatcccggcggcg ccgggcttcgggaactcgacccagatgccgtcgtcggggttcttcaacaccggtgccggc ggcggatcgggcatcggcaacttgggtgcgggcgtgtcgggcctgctcaaccaggccggc gcggggtcactggtggggacactctcggggctgggcaatgccggcaccctggcctcgggt =gtgctgaacstccggcaccgccatctccgggctgttcaa=cgtga'gcacgctggacgccacc accccggcggtgatctcggggttcagcaacctcggcgaccatatgtcgggggtgtccatc gatggcctgatcgcgatcctcaccttcccacctgccgagtccgtgttcgatcagatcatc gacgcggccatcgccgagctgcagcacctcgacatcggcaacgctttggccttgggcaat gtcggcggggtgaacctcggtttggctaacgtcggtgagttcaacctgggtgcgggcaac gtcggcaacatcaacgtcggcgccggcaacctcggcggcagcaacttggggttgggcaac gtcgggaccggcaacctcgggttcggcaacatcggtgccggcaatttcggattcggcaac, gcgggcctgaccgcgggcgcgggggg'cctgggcaatgtggggttgggtaacgccggcagc ggcagctgggggttggccaacgtgggtgtgggcaatatcgggttggccaacaccggcacc=
ggcaacatcgggatcgggctgaccggggactatcggaccgggatcggcggcctgaactcg ggcaccgggaacctcgggttgttcaactcgggcaccggcaacatcgggttcttcaacacc gggaccgggaacttcgggctgttcaactcgggcagttacagcaccggtgtggggaatgcg ggcacggccagcaccgggttgttcaacgcggggaacttcaacaccggtctggccaatgcc ggctcctacaacaccggcagcctcaacgtgggcagcttcaacaccggcggcgtcaacccg ggcaccgtcaacaccggctggttcaacaccggccacaccaacaccggcctgtt=caacacc ggcaacgtcaacaccggcgcgttcaactccggcagcttca.acaacggggcgctgtggacc' ggt.gactaccacgggctggtcggcttctccttcagcatcgacatcgccggcagcaccatg' ctggacctcaacgaaaccctcaacctgggccccatccacatcgagcagatcgacatcccc ggcatgtcgctgttcga'cgtccacgaaatcgtcgagatcggacccttcaccatcccgcag =
gtcgatgttcccgcgataccgctagagatccacgaatcgatccacatggatcccatcgtc ctggtgcccgccaccacaatt'cccgcacagacgagaaccattccgctggacatccccgcc tcacccgggtcaaccatgacgcttccgctcatcagcatgcgcttcgaaggcgaggactgg atcctcgggtcgaccgcggcgattcccaatttcggagaccccttcccggcgcccacccag 'ggcatcacc.attcacaccggccctggccccggaacgaceggcgagctcaagatatctatt ccgggtttcgagattccgcaaatcgctaccacgagattcctgttggacg.tgaacatcagc ggtggtctgccggccttcaccttgttcgcgggtggcctgacgatccccacgaacgccatc ccgttaacgatcgatgcgtccggcgcgctggatccgatcacgattttcccgggtgggtac acgat.cgacccgctgccgctgcacctggcgctgaatctcaccgtgcccgacagcagcatc ccgatcatcgatgtcccgccgacgccagggttcggcaacaccacggcgaccccgtcg.tcg-gggttcttcaactccggcgccggtggggtgtcggggttcggaaacgtcggggtcgaacctg tcgggctggtggaaccaggcggcgagcgcgctggcggggtcgggatcgggggtgttgaat gtcggcacgctgggctcgggtgtgctcaacgtcggctcgggtgtctcggggatctacaac' accagcgtgttgccgctcgggacgccggcggtgctgtcgggcctcggcaacgtcggccat cagctgtcgggcgtgtctgcggccgggaccgcgttgaaccagatccccatcctcaacatc gggttggcggatgtgggcaacttcaacgtcgggttcggcaacgtcggggacgttaacctg ggcgcggccaacctcggtgcgcaaaacctggggctgggcaacgtcggcaccggcaacctc ggcttcgccaacgtcggccacggcaatatcggtttcggcaattcgggtctgaccgccggc gcggccggcctgggcaacacggggttcggcaatgccggcagcgccaactatggtttcgcc aaccagggcgtgcgcaacatcgggttggccaacaccggcaccggcaacatcgggatcggg ctggtgggggacaacctcaccggcatcgggggcctgaactccggtgccggcaatatcggc ttgttcaactccggcaccggcaacatcgggttcttcaactccgggaccggcaacttcggc .atcggtaactcgggcagcttcaacaccggcatcggcaatagcggaacgggcagcactggg ctcttcaatgccggcagcttcaacaccggcgtggccaacgccggcagctacaacaccggc agcttcaatgccggcgacaccaacaccggggggttcaacccgggcaccatcaacaccggc tggttcaacaccggccacaccaataccggcatcgcc.aactcgggcaacgtcggcaccggc gcgttcatgtcgggcaacttcagcaacggcctgttgtggcggggtgatcacgagggcctg ttcagcctgt=tctacagcctcgacgtgccccggatcaccatcgtggacgcccacctcgac ggcggcttcggacccgtggtcctcccgcccatcccggtgccggccgttaatgcgcacctg=
.accggaaacgtcgcgatgggcgcattcaccattccgcagatcgacatccccgcactcacc ccaaacatcaccggaagcgccgccttccgcatcgttgtggggtccgtgcgcattccgccg gtgagtgtcattgtggagcaaataatcaacgcctcggttggggcggagatg=aggatagat cccttcgaaatgt=ggactcaaggcactaatggccttggtataaccttctattcattcgga tcggccgacggttcgccctacgccaccggcccactcgttttcggcgccggcacgagcgac ggaagccatctcaccatttccgcgtccagcggggcgtttaccactccgcagctcgaaact ggcccgatcacgttgggcttccaggtgcccggcagcgtcaacgcgatcaccctcttcccc ggtggtttgacgttcccggcgacctcgctgctgaacctggacgtgaccgccggcgccggc ggcgtggacatcccggccatcacctggcccgagatcgcggcgagcgccgacggctcggtg'-=tatgtcctcgccagcagcatcccgctgatcaacatcccgcccaccccgggCattgggaac =agcaccatcaccccgtcgtcgggcttcttcaacgccggcgcgggcgggggatcgggcttc ggcaacttcggcgcgggcacctcgggctggtggaaccaggcgcacaccgcgctggcgggg gcgggctcgggttttgccaacgttggcacgctgcattccggtgtgctcaacctgggctcg .ggtgtctcggggatctacaacaccagcacgctgggggtggggaccccggcgctggtctca ggcctgggcaacgtcggccaccaactgtcggggctgctttccggcgggtccgcggtgaac ccggtgaccgttctgaatatcgggttggccaacgtcggcagccacaacgccggtttcggc aatgtcggggaggtcaacctgggcgcggccaacctcggcgcgcacaacctgggcttcgga aatatcggcgccggcaacctggggttcggcaatattggccacggcaatgtcggagtcggc aactcgggtctgaccgcgggcgtgccgggcctgggcaatgtggggttgggcaatgccggc ggcaacaactgggggttggccaacgtgggcgtgggcaatatcgggttggccaacaccggc accggcaacattgggatcgggctgaccggcgactaccagaccggcatcggcggcc.taaat tccggtgccggcaacctggggttgttcaactccgg.cgccggcaacgtcggggttcttcaac accgggaccggcaactt'cgggttgttcaactccggcagcttcaacaccggcgtcggcaat agcggaacgggcagcactgggctcttcaatgccggcagtttcaacaccggtgtggccaac gccggcagctacaacacgggcagcttcaatgtcggtgacaccaacaccgggggct'tcaac ccgggcagcatcaacaccggctggctcaacgccggcaacgccaacaccggggtggccaac=
gcgggcaatgtcaacaccggcgccttcgtcaccggcaacttcagcaacggcatcctgtgg' .cgcggcgactaccag=ggcctggccggcttcgecgtgggctacaccctcccgctg.ttcccc gcggtgggcgccgacgtcagcggcgggatcggcccgattaccgtgctgccgcccatccac atcccgcccattccggtcggcttcgccgcggtcggtggcatcggcccgatcgccatcccg gacatctctgttccatccattcac.ttgggcctcgaccccgccgtccatgtcggctccatc accgtcaaccccattaccgtcaggaccccgcccgtgctcgtcagttactcccaaggagcc=
gtcaccagcacgtccggaccaacctcagagatttgggtcaagcccagcttcttccccgga atccggatcgcgccctctagcggcgggggtigcaacgtecacgcaaggggcatactttgtg .gggcccatctccatcccctccggcacggtgaccttcccgggattcaccatcc.ccctcgac ccgatcgacatcggcctgccggtgtcgctgaccatcccggggttcaccatcccgggcggc acectgatccccaccctcccgctgggcctcgcgttg'tccaatggcatcccgcccgtcgac,=
=atc'ccggccatcgttctcgaccggatcttgctggacctgcacgccgacaccactatcggc ccgatcaacgtcccgatcgccgggttcggcggggcgccgggtttcgggaactcgaccacg ctgccgtcgtcgggcttcttc=aacaccggagctggcggcggttcgggctttagcaaca=cc=
ggcgcgggcatgtcgggattgctcaacgcgatgtcggatccgctgctcgggtcggcgtcg ggcttegccaacttcggcacccagctctccggcatcctcaaccgcggcgccggcatctcg ggcgtgtacaacaccggcgcgctgggtgttgtcaccgcggccgtcgtctcgggtttcggc aacgtcggccagcaactgtcgggcttgctcttcaccggcgtcgggccctaa (SEQ ID NO: 26) These polynucleotides include DNA, cDNA and RNA sequences which encode the polypeptide of interest. Silent mutations in the coding sequence result fram the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue. Thus, for example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, .TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC;
aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alaniiie can be encoded by GCT, GCC, GCA; or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be =encoded by TAT or,=TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard.genetic code can,be found in various sources (e.g., L: Stryer, 1988, Biochemistry, 3a Edition, W.H. 5 Freeman.and Co., NY).
~. = =
15. A nucleic acid-encoding an Mtb polypeptide can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase =chain reaction (LCR), the transcription-based amplification system (TAS), the =self-sustained sequence replication system (3SR) and the Q(3 replicase amplificatioxi system (QB). For example, a polynucleotide encoding the protein can be,isolated by polymerase,chain reaction of cDNA using primers based on the DNA sequence of the molecule: A wide variety of cloning and in vitro ampiification methodologies, are well known to persons skilled in the art. PCR methods are described in,=for *
example, U.S. Patent No. 4,683,195; Mullis et al., Cold Spring Harbor Symp.
Quant.

Biol. 51:263, 1987; and Erlich, ed., P.CR Technology; (Stockton Press, NY, 1989).
Polynucleotides also can=be isolated by screening genomic or'cI)NA libraries with probes-selected from the sequences of the desired polyriucleotide under stringent hybridization conditions:
5'- , . . The polynucleotides encoding an Mtb polypeptide include a recombinant DNA which. is-incorporated into a vector into an= atitonomously replicating plasmid or virus or into the geno.mic DNA of a prokaryote 'or eukaryote, *or=' which exists as a=
separate molecule (siich as a-cDNA) independent of other sequences. The nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, 'or modified forms of either nucleotide. The term includes single and double=forms=of DNA. ' = = =
In one embodiment, vectors are used for expression in yeast such as S.
cerevisiae or Kluyveromyces lactis. Several promoters are known to be of use in -yeast-expression systems such as the constitutive promoters plasma niembrane H+-'ATPase. (PMA1), glyceraldehyde-3-phosphate dehydrogenase (GPD), phosphoglycerate kinase=l (PGKI), alcohol dehy,drogenase-1 (ADH1), and pleiotropic drug-resistant pump (PDR5). In addition, may inducible promoters are 6f use, such as GALI-10 (induced by galactose), PHOS (induced by low ==
extracellular inorganic phosphate), and tandem heat shock HSE elements (iriduced.
by temperature elevation to 37 C). Promoters that direct variable expression in response to a titratable inducer include the methionine-responsive MET3 and pronioters.and copper-dependent CUPl promoters. Any of these promoters may be cloned into multicopy (2 ) or single copy (CEN) plasmids to give an additional level of control in expression level. The plasmids can include nutritional markers (such=as 25, URA3, ADE3, HISI, and others) for selection in yeast and antibiotic resistance (AMP) for propagation in bacteria. Plasmids for expression on K lactis are known, -such as pKLAC 1. Thus, in one example, after amplification in bacteria, plasmids cati be iritroduced into the corresponding yeast auxotrophs by methods similar to bacterial transformation. =
:30 -- The Mtb polypeptides can be.expressed in a variety' of yeast strains.
'For example, seven pleiotropic drug-resistant transporters, YOR1, SNQ2, PDR5, YCFI, PDR10, PDRI1; 'and PDRI S, together with their activating transcription factors, PDRI and PDR3, have been simultaneously deleted in yeast host cells, rendering the resultant sfrain sensitive to drugs. = Yeast strains with altered lipid composition of the plasma membrane, such as fhe erg6 mutant defective in ergosterol biosyinthesis, can also be utilized. Proteins that :a're highly sensitive to proteolysis can be expres'sed in 5-. a yeast lacking the' master vacuolar-endopeptidase Pep4, which controls the activation of other vacuolar hydrolases. Heterologous.expression in strains carrying temperature-sensitive (ts) =alleles of genes can be employed if the corresponding null mutant is inviable.
Viral vectors can also be prepared encoding the.Mtb-polypeptides disclosed herein. A number of viral vectois have been constructed, incliuding polyoma, (Madzak et al., 1992, J. Gen. Virol., 73:15331536), adenovirus (Berkner, 1992, Cur. .
Top. Microbiol. Immunol.,'158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Nad.
Acad. Sci. USA, 89:2581-2584; Rosenfeld et a1.,1992; Cell, 68:143-155;
Wilkinson et al.,.1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet et a1.,;1990, -Hum. Gene Ther:, 1:241-256), vaccinia virus (1Vlackett et al., 1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol.
Inununol., 158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses including HS.V and EBV (Mar'golskee, 1992; Curr. Top.. Microbiol. Immunol:, 158:67-90;
Johnson=et al., 1992, J. Virol., 66:2952296.5; Fink et al., 1992, Hum. Gene Ther.
3:11-19; Breakfield et al.; 1987, Mol. Neurobiol., 1:337-371; Fresse et al.,'1990, Biochem. Pharmacol., 40:2189-2199), Sindbis viruses- (H. Herweijer et'al., 1995, Human Gene Therapy. 6:1161-1167; U.S. Patent No. 5,091,309 and U.S. Patent No. 5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol. 11:18-22; I.
Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377) and.retroviruses of =
avian (B'randyopadhyay et al., 1984; Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top. IVlicrobiol.
Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol., -5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol., 54:401-407), and human origin (Page et al., 1990; J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J: Virol., 66:2731-2739). Baculovirus (Autographa californica multinuclear polyhedrosis virus; AcMNPV) vectors are also 'known in the art, and may be obtairied from commercial sources (such as Pha=rMingen, Sa.n'Diego, Calif.;.Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).
Viral vectors, such as po'xviral vectors, that -encode an 'Mtb polypeptide include at least one expression control element operationally linked to the nucleic acid sequence encoding the Mtb polypeptide. The expression c.ontrol'elements are inserted in the viral vector to control and-regulate the expression of=the nucleic acid =
sequence: Examples of expression control elements'of use in these vectors includes, but is not limited to, lac system, operator and prombter regions of phage Iambda, yeast promoters and promoters derived from polyoma, adenovirus; retrovirus or = 10 SV40. Additional operational elements include; but are not limited to, leader sequence, termination codons, polyadenylation signals and any other sequences necessary for, the appropriate transcription. and subsequent translation of the nucleic acid sequence encoding the Mtb polypeptide in the host system. The.expression vector can contain additional elements necessary for the transfer =and subsequent replication of the expression vector containing the nucleic acid sequence in the host.
system. Examples of such elements include, but are not liniited to; origins of replication and selectable markers. It will further.be understood by one skilled in the art that such vectors are easily constructed using conventional methods (Ausubel et al., (1987) in "Current Protocols in Molecular Biology," John Wiley and Sons, New York; N.Y.).and are commercially available. . ==
DNA sequences encoding an Mtb polypeptide can be expressed in vitro by DNA. transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic.
The term also includes any progeny of the subject host cell. ' It'is understood that.all progeny may.not be identical to the parental cell since there may be mutations -that occur during replication. Methods of stable transfer, meaning that the foreigri DNA
is contiriuously maintained in the host, are known in the art. =
As noted above, a polynucleotide sequence encoding an Mtb polypeptide can be operatively linked to expression control sequences. An expression control sequence -operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression.
control sequences. The expression control sequences, include, but are not limited to, appropriate promoters; enhancers, transcription terminators, a start codon (i.e., ATG) : in front of a protein-encoding gene, splicing signal for introns, maiintenance of the.
correct reading frame of 'that gene to permit proper translation of mRNA,.and stop codons.
Hosts cells cari include.microbial; yeast,Ansect and inammalian host cells.
Methods of expressing DNA sequences having eukaryotic or viral sequerices in prokaryotes are well.known.in the art. Non-limiting examples of suitable host cells .
include bacteria, archea, insect, fungi (for example; yeast), mycobacterium (such as M. smegmatis),.plant; and animal.cells (for example, mammalian cells, such as..
human). Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines.
Techniques Tor the propagation of mammalian cells in culture are well-known (see, :
Jakoby and Pastan (eds), 1979, Cell Culture. Methods in Enzyrnology, volume 58, Academic Press, Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly used mammalian host. cell lines are VERO and HeLa cells, CHO cells, and W138, BHK, arid COS cell lines, although cell lines may be used, such as cells designed to provide higher expression desirable glycosylation patterns, or other featu'res. As discussed above, techniques for the trarisformation of yeast cells, such as =
polyethylene.glycol transforrnation, protoplast transformation and gene guns are also known in the art (see Gietz and Woods Methods in Enzymology 350: 87-96, 2002).
Transformation of a host cell.with recombiriant DNA can .be carried out by conventional'techniques as are well knowri to those skilled in the art. Where the host is prokaryotic, such as, but not limited to, E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential ' growth phase and subsequently treated by the CaC12 method using procedures well known in the art. Alternatively, MgC12 or RbCI can be used. Transformation can also be performed after forming a protoplast of the host cell.if=desired, or by electroporation. .
When the host is a eukaryote, such methods of transfection of DNA as -. calcium phosphate coprecipitates, conventio.nal n=iechanical procedures such as microirijection, electroporation, insertion of a plasmid encased in liposomes;
or virus vectors can be used. Eukaryotic cells can also be co-transformed with -48= . = . ...
polynucleotide sequences encoding an.Mtb polypeptide, ancl a second foreign DNA
molecule encoding a'selectable phenotype, such as the herpes simplex thymidirie kinase gene. = Ariother method is to use a,eukaryotic viral vector, such as simian virus 40 (SV40)"or bovine papill'oina virus, to t'ransiently iiifect or transform 5= eukaryotic cells and express the.protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). = .

Methodfor Detecting an Mtb Infection: Detection of T Cells, Methods for detection of a Mycobacterium infection in a subject are 10' disclosed herein. In several embodinients, a Mycobacterium infection can be detected based 6n the presence of T cells in a biological sample, wherein the T cells specifically react with.a Mtb polypeptide'disclosed herein (see above).
In several embodiments, a biological sample comprising T cells is obtained from s subject of interest. Suitable biological samples include, but are not limited to, 15. blood samples, peripheral blood mononuclear cells, sputum, saliva, -cerebral spinal fluid or samples of isolated T cells (such as CD8+=T cells and/or CD4+
T'aells), lymph node tissue, lung tissue, or other tissue sample. -In one example, the sample is incubated with a Mycobacteriiim polypeptide, as disclosed herein; a polynucleotide encodirig the Mtb polypeptide and an APC that expresses the Mtb polypeptide or a 20 - fragment thereof that binds MHC. The preserice or absence of specific activation of the T cells is detected. The CD8+ T cells and/or CD4+ T cells which recognize the peptide in the detection method have generally been presensitized fn vivo to the Mtb polypeptide of interest. In several embodiments, these antigen-experienced T cells are generally .25. present in the peripheral blood of a host'which has been exposed to the antigen at a frequency of 1 to 106 to 1 in 103 peripheral blood mononuclear cells (PSMCs).
'In one example, the sample is isolated T cells. For example, T cells can'be isolated from a subject of interest by routine techniques (such as by FicolUHypaque density gradient centrifugation of peripheral blood lymphocytes, or by fluorescence = 30 activated cell sorting). In one embodiment the T cells used in the assay are in the form of unprocessed or diluted samples, or are freshly isolated T cells (such as in the form of freshly isolated mononuclear cells (MCs) or peripheral blood mononuclear cells=(PBMCs) which are-used directly.ex vivo, such that ttiey- are not cultured' before being used in -the method. However the 'T cells .can be culttired:
before use, . for exainple in 'the presence of one or more of the peptides, and' generally also =
exogenous growth=prorriotirig cytokines. During culturing tlie.peptides are typically.
presented on the surface of cells such as APCs. Pre-culturing of the T cells may lead to an increase in the sensitivity of the niethod. Thus the T. cells cari be coriverted into cell lines, such as:short term cell lines.
In several embodiments, the T cells are incubated in vitro for two to nine days, such as about four days, at 37 C with an Mtb polypeptide or fragment thereof that binds MHC. In several examples, the Mtb polypeptide or fragment thereof that binds MHC is included (at a concentration of, for example, about 5 to about 25 g/ml, such as about 5, about 10, about 15, or about 20 g/ml). In several examples, another aliquot of a T cell sample can be incubated in the absence of the Mtb polypeptide as a control.
-. In one embodiment, mononuclear cells (MCs) are separated from the sample.
The MCs include the T cells and antigen presenting cells (APCs). Thus in the method the APCs present in the separated MCs can present the peptide to the.T
cells. In another embodiment only T cells, such as only CD8+ T cells, only CD4+ T
cells, or orily CD3+ T cells, can be purified from the sample.
' The APC used in the method may be any cell which has MHC-class I*
molecules on its surface. It may or may not be a specialized antigen presenting cell, stuch as a B cell, dendritic cell or macrophage. The APC used in'the method may be fromthe same host as the T cell. Generally, the APC is capable of presenting the peptide to a T cell. The APC can be a freshly isolated ex vivo cell or a cultured cell 25= such as a cell from of a cell line. =
T cells derived from the sample from the.subject of interest can be.placed.
into an assay with all the Mtb polypeptides (or a pool of the Mtb polypeptides, or a specific Mtb polypeptide) which it is intended to test the relevant panel or the T cells can be divided and placed into separate assays each of which contain one or more of the peptides.' In one embodiment, one or more of the polypeptides with an amino acid sequence set forth as SEQ ID NOs: 1-12, or an fragment of one or more 'of these polypeptides that bind MHC, is utilized. Two or more of any of the Mtb peptides disclosed herein can be used for simultaneous, separate *or.sequential use of T cells thatrecognize these polypeptides: Additional combinations of any of the.
Mtb polypeptides disclosed herein can be utilized.
In one-embodiment the one or more peptide(s) is '(are) provided to the presenting cell in the absence of the T'cell: This cell is then provided to T
cells isolated from the subject, typically after being allowed =to present the peptide on its surface.
The-duration for which the peptide is contacted with the cells will vary depending on the method used for determining recognition of the peptide.
Typically 105 to .107, such as 5 X 10$ to 106 PBMCs are added to each. assay. In the case, vvhere -peptide is added directly to the assay its concentiation is typically from 10"I to =103 g/ml, such as about 0.5 to about 50 gg/ml qr about 1 to aboutl.0 g/m1. The length of time for which the T cells are incubated with the -peptide can be from about 4 to about 24 hours; such as from about 6 to about 16 hours, or for about 12 hours.
The determination of the specific recognition of the-peptide by the T cells can be done by measuring the binding of the peptide to the T cells. Typically T cells which bind the peptide can be sorted based on this binding, for example using a fluorescence activated cell sorting (FACS) technique. The detection of the presence of T cells which recognize the peptide will be deemed to occur if the frequency of cells sorted using the peptide is= above a control value.
Determination of whether the T cells recognize the peptide can also be done by detecting a change in the state of the'T cells in the presence of the peptide or determiiiing whether the T cells bind the peptide: The change in state is generally caused by antigen specific functional activity of the T cell after the T cell receptor binds the peptide. Generally when binding the T cell receptor the peptide is bound=
to an MHC class I molecule, which may be present on the surface of a PBMC or an antigen presentirig cell (APC).
=T cell activation.can be detected by any means known to 'one of skill in the art. In one example, CD8+ T cell activation is detected by e.valuating cytolytic 30 activity. In another example, CD8+ T cell =activation and/or CD4' T cell activation is detected by proliferation. In several examples, a level of proliferation that is at least two fold greater and/or'a level of cytolytic activity that is at least 20%
greater than in uninfected subjects indicates the presence of a Mycobacterium infection in the==
subject of interest:
The change in.state of the T cell may be the starl'of or.increase.in secretion bf a substance froin the T cell, such as a cytokine, such as interferon (IFN)-y, IL-2 or TNF-a. In one example, the substarice can be detected by allowing it. to bind to a specific binding agent'and then measuring the presence of the specific binding .
agent/substance complex. The specific binding agent is typically an antiliody., such as polyclonal or monoclonal 'antibodies that binds the substance, 'such as the '-cytokine. Antibodies to cytokines are commercially available, or can be made using standard techniques. -Typically the specific binding agent such as the antibody is immobilized on a solid support. After =the cytokine is allowed to bind the solid support can optionally be washed to remove material which is not specifically bound to the antibody.
The antibody/cytokine 'complex cari be detected by using a second binding agent which will bind the complex, such as an antibody that is labeled (either directly or indirectly) with a label. Generally,'the second agent binds the substance at a site which is differerit from the site which binds the first agent.
In several=examples, the second binding agent can'be detected by a third agent which is labeled directlyor indirectly by a detectable label. For example the . second agent may include a biotin, allowing detection by a third agent which comprises a strepavidin and a label, such as an enzymatic, radioactive or fluorescent label.
In one embodiment the detection system is an ELISPOT assay, such as the assay described in.PCT Publication No. WO 98/23960, incorporated herein by reference. In one example, IFN-y secreted from the T cell is bound by a first IFNy specific antibody which is immobilized on a solid support. The bound IFN-y is then detected= using a second IFN-y specific antibody which is labeled with a detectable label. Exemplary labeled antibodies are commercially available,= such as from MABTECHTM (Stockholm, Sweden). An exemplary ELISPOT assay is described in the Examples section below. = = =
'The change in state of the T cell also can be measured may be the-increase in the uptake of substances by the T cell, such as the uptake of thymidine: The change .

= = .=... . = . . . .= =. = .= = = . , .in state can also be measured by an increase in the size of the T cells, or proliferation of the T cells, or a change=in cell surface markers on the T cell.
Reagents are provided herein for the detection of CD8 expressing cells (CD8+) that specif cally bind an Mtb=polypeptide as disclosed herein. ==These reageiits are tetrameric.MHC =Class I/immunogenic TARP polypeptide complexes.
These tetraixieric complexes include an Mtb polypeptide, si.ich as a polypeptide of =
nine to twenty amino acids in length that specifically binds MHC class I. =
Tetrameric MHC Class I/peptide complexes can be syntliesized using methods well knowri in the art (Altmann et a1.,.Science 274:94, 1996, which is herein incorporated by reference). In rion-limiting example, purified HLA heavy chain polypeptide and B2-microglobulin (B2m) can be synthesized by means of a prokaryotic expression system. One specific, non-limitirig example of an expr'ession system.of use is the pET system (R&D Systems, Minneapolis,lV1N):
The heavy chain is modified by deletion of the trans-membrane and cytosolic tail and 'COOH-terminal addition of a sequence containing the biotin protein ligase (Bir-A) enzymatic biotinylation site. Heavy chain,132m, and peptide are then refolded.
The refolded product can be isolated by any means known in the art, and then :'biotinylated by Bir-A. A tetramer is then produced by contacting the biotinylated product with strepavidin. .
In one embodiment, the strepavidin is labeled. 'Suitable labels include, but are not limited to, enzymes, magnetic beads, colloidal magnetic beads, hapteris, fluorochromes, metal compounds, radioactive compounds or drugs. The enzymes that can be conjugated to strepavidin include, but are not limited to, alkaline phosphatase, peroxidase, urease and f3-galactosidase. Tlie fluorochromes that can be = conjugated to the strepavidin include, but are not limited to,:fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin, .
allophycocyanins and Texas Red. For additional fluorochrome=s that cari be conjugated to strepavidin, see Haugland, R. P., Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals (1992-1994). The metal compounds that can be conjugated to the strepavidin include, but are not limited to, ferritin, colloidal gold, and particularly, colloidal superparamagnetic beads. The haptens that can be conjugated to the strepavidin include, but are not limited to, biotin, -digoxigenin, oxazalone, and nitrophenol. The- radioactive compounds that caxi.be conjugated to strepavidin are known to the art, and include biut are not limited to technetium 99m (99 Tc), 125 I and amino acids comprising any radionuclides, including; but not limited to, 14 C, 3 H and 35 S. Gener.ally,, strepavidin=
labeleii witfi a fluorochrome is utilizedin the methods disclosed herein.
In one embodiment, susperision of cells iricluding T cells.that specifically.
recogni.ze an Mtb polypeptide is produced, and the.cells ar=e reacted with the tetramer in'suspension. In one embodiment, these reagents are used to label cells, which are then analyzed by fluorescence activated cell sorting (FACS). A
machine -for=FACS employs a plurality of color channels, low angle.and obtuse light-scattering detection channels, and impedance channels, among other more.
sophisticated'levels of detection, to separate or sort cells. Any FACS
technique can ..be employed= as long as it is not detrimental to the detection of the desired cells.
(For. exemplary methods of FACS see U.S. Patent No. 5, 061,620, incorporated herein by reference). .
Method for Detecting an Mtb Infectian: Skin Tests In another aspect, this invention provides methods for using one or more of the polypeptides clescribed above to diagnose Mycobacterium infection, and in.
' partici,ilar tuberculosis, using a skin test. A "skin test" is any assay performed directly. on a patient in which a delayed-type hypersensitivity (DTH) reaction (such a's induration, swelling, reddening or.dermatitis) is measured'following.
administration into the skin, such as the intradermal injection of oine or more polypeptides described above. Such injection can be achieved using any suitable 25. device sufficient to contact the polypeptide or polypeptides with dermal cells of the patient, such as a ttiberculin syringe or 1 ml syringe. In several examples, the reaction is measured at least 48 hours after injection, such as=between aboiut 48 and about.72 hours after injection. A DTH reaction is a cell-mediated immune response which is greater in subjects that have been exposed previously to the test aritigen'(the Mtb polypeptide, fragment thereof that binds MHC, or fusion protein thereof). The response can be=
measured visually, such as using a ruler. In several examples, a response that is -54- .
:= greater than about 0.5 cm in diameter, such as: greater'=than about 1.0 cm in diameter, is a positive response, and is indicative of Mycobacterium 'infection.
The Mtb polypeptides disclosed herein can be forinulated for use in a skin test as pharmaceutical compositions containing.a polypeptide and a.
physiologically 5. acceptable carrier. These compositions typically contain one o'r more of the disclosed Mtb polypeptides (or a fragment.thereof that binds MHC or=a fusion protein thereof) in an amount ranging from:about T g,.to about 100 g, such as from about 10 gg to about 50 g'in a:volume of 0.1 ml:-'The carrier employed in a pharmaceutical composition can be a saline solution with appropriate 'preservatives, such as phenol and/or T=WEEN80TM. . Generally, the polypeptide employed in a skin test is of sufficient size siich that it remains at the site of =injection for the duration of the'reaction period. In several examples, a polypeptide that is at least nine amino acids in length'is ' sufficient. Without being bound by theory, the polypeptide is broken down by macrophages within hours of injection to allow presentation to T-cells: Such polypeptides can contain repeats=of one or more of the above disclosed sequences and/or other.immunogenic or non-immunogenic sequences. ==
Thus, the determination of the recognition of the peptide by the T cells can be measured in vivo. In several examples, the .peptide is administered to the - individual and then a response which indicates.recognition of=tlie peptide may be =
measured. In one embodiment the peptide is administered intradermally, typically in a similar rrianner to the Mantoux test. The peptide can' be administered .epidermally.
The peptide is typically administered by needle, such as by injection, but can be administered by other methods such as ballistics, for example the ballistics techniques which have been used to deliver nucleic acids. Published EPC
Application No. EP-A-0693119 describes techniques which can typically be used to administer the peptide. In several examples, from '0.001 to 1000 - g, for example from 0.01 to 100 gg or 0.1 to 10 g of peptide is =administered. Alternatively an agent can be administered which is capable of providing the peptides in vivo.
Thus a . polynucleotide capable of expressing the polypeptide can be administered.
The polynucleotide typically has any of the characteristics of the polynucleotide which is discussed below. Polypeptide is expressed from the polynucleotide in vivo aind recognition of the peptide- in vivo may. be measured. Typically from 0.001to g, for example frorn Ø01. tb 100 g or 0.1 to 10 g of polynucleotide is =.
= administered.

Methodfor Detecting an Mtb Infection: Detection ofAntib'odies.
Methods are disclosed herein wherein the polypeptides described above are used to diagnose Mycobacterium infection, and in particular tuberculosis., In these embodiments, methods are'provided for detecting Mycobacterium infection'in a biological sample,'using one or more of the above polypeptides, alone or in combination. In several embodiments.in multiple polypeptides are employed. The polypeptide(s) are used in.an assay to determine the presence or absence of antibodies to the polypeptide(s) in a biological sample (such as, but not limited to, whole blood, sputum, serum, plasma, saliva, or cerebrospinal fluid) relat'ive to a control. The presence of such'antibodies iridicates previous sensitization to mycobacterial antigens which may be indicative of Mycobacterium infection, arid in particular tuberculosis. . ' In embodiments in which more than one polypeptide is employed, the polypeptides can be complementary, such that one component polypeptide will detect.infection in samples where the infection would not be detected by ariother component polypeptide). Complementary polypeptides.may generally be identified by using each polypeptide individually to evaluate serum samples obtained from a series of patients known to be infected with Mycobacterium. After determining which samples are correctly identified as,positive with each polypeptide, combinations of two or more polypeptides may be formulated that are capable of detecting infection in most, or all, of the samples tested. Complementary polypeptides are of use to improve sensitivity of a diagnostic test. Thus, more than one of the above-described Mtb polypeptides can be included in an.assay:
Additional polypeptides from Mtb (those inot described here'in) optionally can be included in the assay. . .
There are a variety of assay forrnats that can be used to detect antibodies in a sample (see, for example, Harlow and Lane; Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988), which is incorporated herein by reference).
In -56= = .

general, the presence or absence of an Mtb infection in a patient may be determined by (a) contactirig a biological sample obtained from=a patient with one or more Mtb polypeptides; (b) detecting in the sample the presence (or absence) of.an antibody that =binds to.the polypeptide(s); and (c) comparing the.level of antibody with a5 control. The control can be a standard value, such as =a pre-determihed ci.ut-off ' value. The control'can be the amount of antibodies in a subject known to be infected with Mtb, or the amount of antibodies that specifically bind the polypeptide(s) in a subject known not to be infected=with Mtb. = .
-In several embodiments, the assay involves the use of a polypeptide immobilized on a solid support. Antibodies that =specifically bind the polypeptide(s) of interest bind to the solid support. The bound antibody can then be detected using a detection reagent that includes a detectable label. Suitable =detection reagents include labeled antibodies that bind to the antibody/polypeptide complex.
Suitable detection reagents also include second unlabeled antibodies that bind to-the antibody polypeptide complex and a third antibody that specifically binds the second antibody. Suitable detection reagents also include unbound polypeptide labeled with a reporter group (such as in a semi-competitive assay). Alternatively, a competitive assay may be utilized, in which an antibody that binds to the potypeptide of interest is labeled with a reporter group is incubated with the= sainple: Following incubation, the antibody is then allowed to bind to the immobilized antigen after incubation of the antigen with the sample. The extent to which components of the sample inh'ibit the binding of the labeled antibody to the immobilized polypeptide is indicative of the reactivity of the sample ~with'the immobilized polypeptide. = . .
.= A solid support used in an assay disclosed. herein can be any solid material to which the antigen may be attached.. For example, the solid support can be a test well in =a microtiter =plate or a nitrocellulose or other suitable membrane.
Alterinatively, the solid support may be a bead or disc, such as glass, fiberglass, =latex or a plastic material such as polystyrene or polyvinylchloride. The support can also be a magnetic particle or a fiber optic sensor, such as those disclosed, for exarriple, iri U.S. Pat. No. 5,359,681. ' The polypeptides can be bound to the soli=d. support using a variety of techniques. The'binding of the polypeptides can be accoinplished by a noncovalent association, such as adsorption, or covalent attachment, such' as a direct liinkage between the antigen and functional groups on the support or a linkage through a cross-linking agent.
For binding by adsorption, binding can be achieved by corntacting one or more Mtb polypeptide(s) (generally in a buffer) ~ivith the solid support for a suitable amount of time. The contact time for. binding is typically between about 1=hour and '1 day. In general, binding is achieved by'contacting- a polystyrene or 10. polyvinylchloride solid support with an amount of the one or more Mtb polypeptide(s) ranging from about 10 ng to about 1 g, such'as about 100 rig of antigen. . ' ."
Covalent attachment of the Mtb polypeptide(s) of interest to a solid support can. generally be achieved by reacting the support with a bifunctional reagent that reacts witli both the supporE and a functional group, siich as a hydroxyl or amino group, on the polypeptide. For example, an Mtb polypeptide can be bound to=
supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active -hydrogen on the polypeptide (Pierce Immunotechnology Catalog and Handbook, at .20 A12 A13, 1991). =
In certain embodiments, the assay is an enzyme linked immunosorbent assay (ELISA). This assay can be performed by first contacting a polypeptide antigen that has been immobilized on a solid support'(such as in the well of a microtiter=plate) with the sample in a manner such that that antibodies present within the sample that . 25 specifically bind the polypeptide of interest bi:nd the immobilized polypeptide.
- Unbound sample is then removed and a detection reagent capable of binding to the immobilized antibody-polypeptide complex is added. The amount of detection =
reagent that.remains bound is determined using a method appropriate for the specific detection reagent. For example, the detection method can detect fluorescence or the 30 presence of an enzymatic activity. =
In some embodiments, the polypeptide is immobilized ori the.support; any remaining protein binding sites on the support are typically blocked. Any suitable -58-=
blocking agent can be 'used to block the unbound proteiri binding sites, such as.
bovine'serum albumin or TWEEN 20TM can be employed. The immobilized polypeptide is then incubated with the sample, aind the antibody is allowed to bind to the antigen. The sample -can be diluted with a suitable diluent, for example a.buffer 5= such'as phosphate-buffered saline (PBS) prior to incubation. In. general, an appropriate contact-time (incubation tirne) is a period of time that is sufficient to detect the=.presence of antibody in a Mycobacterium-infected sample. In one speci f c, non-limiting example, the contact timeis sufficient to achieve a level'of binding that is at least 95% of that achieved'at equilibrium between 'bound and, unbound antibody. The time necessary to achieve equilibrium can be determined by assaying the level.of binding that occurs over a period of time. At room' temperature, an incubation time of about 30 minutes is generally sufficient.
Unbound sample can then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% TWEEN 20TM. A detection =15 reagent can then be added to the solid support. A detection reagent canbe any corripound that binds to the immobilized antibody-polypeptide.complex and can be detected. In'several embodiments, the detection reagent contains a binding agent :
(such as, for example, Protein A, Protein G, immunoglobulin, lectiri or free antigen) conjugated to a label. .Labels of use include enzymes (such as horseradish peiroxidase), substrates, cofactors; inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. The conjugation .of a binding agent to a label can be achieved using methods known in the art; conjugated binding agents are also coniriiercially available (such as from Zymed Laboratories, San Francisco,-Calif., and Pierce, Rockford, Ill.). . ' 25 . The detection reagent is incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect.the bound antibody.
An appropriate amourit of time may generally be determined from the manufacturer's instructions or. by assaying the level -of binding that occurs over a period of time. Unbound detection reagent is then removed and bo.urfd detection reagent *is detected using the label. For radioactive labels, scintillation counting or autoradiographic methods can be used for detection. Spect=roscopic methods may be .used to detect dyes, luminescent groups and fluorescent groups used as labels.

Biotin'can be detected using avidin coupled to a different label, stich as a radioactive label, fluorescent label or an enzymatic label. Enzymatic= labels can be detectecl by the addition of substrate (generally for a specific period of time), follovved by spectroscopic or otlier= analysis of the reaction products.*
To determine the preseince or absence of anti-Mycobacterium antibodies in the sample, the signal detected from the label that bound to the solid support'is :
generally compared to a control. In. one embodiment, the coritrol is a standard value, such as tlie= average mean signal obtained when the immobilized antigen is incubated with samples from an uninfected patient. In general, a, sample generating a signal = Ihat is two or three standard deviations above the control is considered positive for Mycobacterium infection. In another embodiment, the control value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co.,pp.
106 107 (1985). 'Briefly, in this embodiment, the control value is determined from a plot of pairs of true positive. rates (sensitivity) and false positive rates (100%
specificity) that correspond to each pcissible coritrol value for the diagnostic test result. = The control value on the plot that encloses the largest area is the mo'st accurate cut-off value, and a sample generating a signal that is higher than the cut-.
off value determined by this method is considered positive. Alternatively, the cut-off value may be shifted to minimize the false positive rate, =or to minimize the false negative rate. In general, a sample generating a signal that is higher than, the cut-off value deterrriined by this metliod is considered positive for tuberculosis:
In a related embodiment, the assay is perforined in a rapid flow-through or =
strip test format, wherein the antigen is immobilized on a membrane; such as, but not=liniited=to, nitrocellulose. In a flow-through test, antibodies within the sample bind to the immobilized'polypeptide as the 'sample passes through the membrane. A
detection reagent (for example, protein A-colloidal gold) binds to the antibody-polypeptide complex as the solution containing the detection reagent flows through the membrane. The detection of bound detection reagent can be performed as 30. described above. . .
In one example of the. strip test format, one end of the membrane to which the polypeptide is bound is immersed in a solution containing the sainple.' The sample migrates al.ong the membrane through a region containirig the detection reagent and, to the area of immobilized polypeptide. The concentration of the detection reagent at the polypeptide indicates the presence of anti-Mycobacterium -=
antibodies in the *sample. Typically, the concentration of detection reagent at that 5 :site.generates a pattern, such as a line, that can be read visually: The absence of such a pattern indicates a negative result: In general, the amount:of=polypeptide immobilized on the membrane is'selected to generate a visually discernible patterri when the biological sample contains a level -of antibodies that would be.
sufficient to generate a positive signal in an enzyme linked immunosorbaint assay (ELISA). -In several embodiments, the amount of polypeptide immobilized on the membrane ranges from aboiit 25 ng to about 1 g, such as from about 50 ng to about 500 ng.
Such tests can typically be performed with a very small volume of patient serum or blood.

MetFcod for Detecting an Mtb Infection: Detection of Pol,ynucleotides Diagnostic reagents'include the use of polynucleotide sequences encoding one or more of the above disclosed Mtb polypeptides. Mycobacterium infection can be detected by detecting the presence, absence, or level.of mRNA encoding a .Mycobacterium polypeptide in a biological sample. In several examples,~
20. hybridizatiori assays are utilized, such as Northern blot or dot blot assays. In additional e-kamples, PCR based assays are utilized.
General methods for mRNA' extraction are well known in the art'and are disclosed in standard textbooks of molecular b'iology, including Ausubel et al., Current.Protocols of Molecular Biology, John Wiley and Sons. (1997). Methods fo'r RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andres et al., BioTechniques 18:42,044 (1995). In particular, RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as QIAGEN , according to the manufacturer's instructions. For example, total RNA from cells in culture (such as -those obtained from a subject) can be isolated using QIAGEN
RNeasy mini-columns. Other commercially available RNA isolation kits include MASTERPt7RE . Complete DNA and RNA Purification Kit (EPICENTRE ' Madison, Wis.), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Totai RNA
from tissiue samples can be-isolated using RNA Stat-60 (Tel-Test). RNA
prepared a biological sample can be isolated, for example, by. cesium chloride density gradient centrifugation. . ;
.5 . Methods for quantitating mRNA are well known in the art. In one example, the method utilizes reverse transcriptase polymerase chain reaction (RT-PCR).
Generally, the first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template irito eDNA, followed by.its exponential amplification in a PCR reaction. The two most commonly used reverse 10. transcriptases are avian -myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be.reverse-transcribed usirig a GeneAmp 15 RNA PCR kit (Perkin Elmer, Calif., USA), following th.e manufacturer's instructions.. The derived cDNA can then be used as a template in the subsequent PCR reaction. Although the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically .employs the Taq DNA =polymerase, which has a 5'-3' 20 nuclease activity but lacks a 3'-5' proofreading endonuclease activity.
Thus, TaqlVlan PCR typically utilizes the 5'-nuclease activity of Taq or Tth polymerase to' hydrolyze'a hybridization probe bound to its target ainplicon, but any enzyme with equivalent 5' nuclease activity can be used. Two oligonucleotide, prirners are used to generate an aniplicon typical of a PCR reaction. A third oligonucleotide; or 25 probe; is designed to detect nucleotide seqiience located between the two PCR
primers: The probe is-non-extendible by Taq DNA polymerase enzyme, and' is ' labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-, induced emission from the reporter. dye is quenched by the quenching dye when the two dyes are 'located close together as they are on the probe. During the =
30 amplificatiori reaction, the Taq DNA'polymerase enzyme cleaves the probe in a.
template-dependent manner. The resultant probe fragments disassociate.in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule.of reporter -dye. is' liberated for each new molecule synthesized, and detection of.the unqueinched'reporter dye provides tlie ba=sis for quantitative interpretation of the data.
TAQMAN RT-PCR can be=performed using commercially available equipment, such as, for example; ABI PRISM 7700 Sequence Detection System.TM. (Perkin-Elmer-Applied Biosystems, Fpster City, Calif., USA), or Lightcycler (RoclieMolecul'ar Biochemicals; Mannheim,. Gerinany). In one.
embodiment, the 5' riuclease procedure is run on a real-time quantitative PCR-device such as the.ABI PRISM 7700 . Sequence Detection S.ystem . The system iricludes of therinocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During .
amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD: 'The system includes software for. runining the.instrument and for analyzing the data.
In some examples, 5'-Nuclease assay data are initially expressed as Ct; or the ' threshold cycle. As discussed above, fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cyycle (Ct).
To minimize errors and.the effect of sample-to-sample variation,.RT-PCR is . can be performed using an intemal standard. The ideal internal standard=is expressed at a constant level among different tissues, and is unaffected by the experirriental treatrnent. RNAs most frequently used to normalize=patterns of.gene expression are mRNAs *for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase-(GAPDH), beta-actin, and 18S ribosomal RNA.
A..more recent variation of the RT-PCR techniqiue is the real time =
quantitative PCR, which measures PCR product accumulation through a dual- ' labeled fluorigenic probe (i:e., TAQMAN probe). Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR.using a normalization gene contained within the sample, or a housekeeping gene for.RT-PCR (see Held et al., Genome Research 6:98'6 994, 1996). Quantitative PCR
is.also -63- .

described in U.S. Pat. No. 5,558,848, the disclosure of wliich- is incorporated.herein :by reference. = Related probes and quantitative aniplification piocedures are described in U.S. Pat..No. 5,716,784 and U.S. Pat. No. 5,723,591, the disclosures of which are incorporated herein by i=eference. Instruments for carrying.out ' quantitative PCR. in microtiter plates are available. from PE Applied Biosystems, 850 Lincoln=Centre Drive, Fcisfer City, Calif. 94404 under the trademark=ABI PRISM

7700.
The steps of a representative protocol for quantitating gene expression using.
fixed, paraffin-embedded tissues as the RNA source, including mRNA isolation, purification,. primer extension and amplification are given in various published journal articles (see Godfrey et al. J. Molec. Diagnostics 2: 84 91, 2000; K.
Specht et al., Am. J. Pathol. 158: 419 29, 2001). Briefly, a representative process starts with cutting aboitt 10 m thick sections of paraffin-embedded tissue sample. The RNA is then extracted,.and protein and DNA are removed. After analysis of the RNA
concentration, RNA repair and/or amplification= steps can be included, if necessary, and RNA is reverse transcribed using gene specific promoters followed by RT=PCR.
An alternative quantitative nucleic acid amplification procedure is described in U.S. Pat. No. 5,219,727, which is incorporated herein by reference. In this procedure, the amount of a target sequence in a sari=iple is determined by simultaneously aniplifying the target sequence and an internal standaid'riucleic acid segment. The amount of amplified DNA from each segment is determined and compared to a standard curve to determine the amount of the target nucleic acid=
segment that was present -in the sample prior to amplificati6n: In some embodimeints of this method, the eXpression'of a "house keeping"

gene or "internal control" can also be evaluated. These terms are 'meant to iriclude any constitutively or globally expressed gene whose presence enables an assessment' of cytokirie mRNA levels. Such an assessment comprises a determination of the overall constitutive level of gene transcription and a control,for variations in RNA.
recovery.

Monitoring the Progression of an Infectioiz and/or Effectiveness of Therapy.
.=
In several embodimerits, the diagnostic=methods=disclosed herein are used for monitoring the'progression of a Mycobacterium infection.. In this embodiment,' assays' as described above for the diagnosis of a= Mycobacteriuin irifection may be 5'. performed over. time, and the change iri the level 'of react'ive polypeptide(s) -evaluated. For example, the assays can be performed about every 12, 24, 36, 48, 60 or 72 hours for a specified period, such as over months- or weeks, :and thereafter performed as needed.
In some examples, the presence of an Mtb polypeptide, or a polynucleotide encoding.the polypeptide is assessed. Generally, the Mycobacterium infection is progressing in those patients in whom the level of polypeptide (such as detected using a binding agent), the level of polynucleotide, the level of antibodies, or the level of T cells increases over time. In contrast, the Mycobacterium infection is= not progressing when the level-of reactive polypeptide, the level of polynucleotide, the level of antibodies, or the level -of T cells either remains constant= or decreases with.
time. In this manner, the effectiveness of a particular therapeutic regimen can be assessed. In one enibodiment, the presence of an Mtb polypeptide is assessed in a subject.. The subject is administered a therapeutic protocol. The presence of the Mtb polypeptide is then assessed. An increase or no change in the amouint of the Mtb polypeptide (or polynucleotide) as compared to the amount of the Mtb polypeptide prior to the administration of the therapeutic protocol indicates that the =therapetitic protocol in not effective, and the Mtb infection is progressing:' A
decrease in the amount of the Mtb polypeptide (or polynucleotide) as compared to the amount, of the Mtb polypeptide (or polynucleotide) prior to the administration of the therapeutic protocol indicates that the therapeutic. protocol is effective, and that the Mtb infection is not progressing.
In another embodiment, the presence-of T cells, such as CD8+ T cells and/or CD4+ T cells, that specifically recognize an Mtb. polypeptide' is assessed in a subject.
'I"he subject is administered a therapeutic protocol. 'The presence of the T
cells that specifically recognize the Mtb polyp.eptide is then assessed. Ain decrease or:
no ' change in the amount of CD8+=T cells and/or CD4+ T cells that specifically recognize the Mtb polypeptide as compared to the amount of the CDB+ T'cells and/or CD4+ T cells; respectively, that specifically recognize the Mtb polypeptide prior to the administratiori of the therapeutic protocol indicates that the therapeutic protocol in not effective. An increase in the amount of the CD8+ T cells and/or CD4+ T cells'that specifically recognize the Mtb polypeptide- as compared.to the amount of the CD8+T cells and/or CD4+ T cells that specifically recognize the Mtb polypeptide prior to the administration of the therapeutic p'rotocol indicates that the therapeutic protocol is effective.
It should be noted that for. any, of the above-described assays, to improve sensitivity, multiple Mycobacterium markers may be assayed within= a given sample.
It will be apparent that the assays disclosed herein can be used in combination.
Thus, sets of Mycobacterium polypeptides, and combinations of assays can be for optimal sensitivity and specificity. Numerous other assay protocols exist that are suitable for use with the polypeptides of the present invention.'The above descriptions are intended to be exemplary only: -The disclosure is illustrated .by the following non-limiting Examples.
EXAMPLES =
For many infections, the repertoire of the CD8 response is shaped by the entry of antigen into the. MHC-I processing pathway, binding of peptides and/or ' non-peptide antigens.to MHC-I molecules, and recognition of these structures by T
cells. Ultiniately, a relatively limited subset of pathogen-specific T cells emerge.
While a number of commonly recognized CD4 Mtb antigens have been described (Reed et a1.,.Microbes Infe.et 7:922-931, 2005) (ESAT-6, CFP10, Ag85, etc.), surprisingly.little is known about common Mtb antigens r.ecognized by human CD8"
T cells. The majority of CD8 epitopes that have been identified were defined by testing of Mtb peptides selected for high affinity binding to MHC Class Ia molecules (HLA-A2 in most cases (see, for example, Lalvani, Microbes Infect 7:922-931, 1998)). In almost all of these, however,= the ex vivo frequency of these T
cells in Mtb-infected individuals is low or undetectable, suggesting that these=specificities may not represent immunodominant responses. : Iri contrast,: in'the 'limited cases. in which T cells have been used to define epitopes contained.in selected Mtb antigens, high ex, vivo'frequencies have=been demonstrated (see Lewinsohn' et al., Am JRespir Crit Care Med 166:843-848, 2002); suggesfing; -that a. T cell-centered approach can 5. identify imrriunodominant epitopes. Moreover, CD8 T cell responses to some Mtb antigens which represent good CD4 antigens (CFP10;.ESAT-6, Ag85,'and 1VItb39), have been detected at high frequency in persons infected with =Mtb. Therefore, a limited library of overlapping synthetic peptides representing several known Mtb antigens was used to determine the magnitude of the CD8 response"to these 'antigens in persons with active tuberculosis (TB) and latent tuberculosis infeotion (LTBI) as well as uninfected subjects. Furthermore, a panel of Mtb-specific.CD8i' T
cell clones was utilized to define minimal epitopes recognized within these anti.gens and determined the contribution of these novel epitopes to the ex vivo Mtb-specific CD8 response. . =
Example 1 .
Materials and Methods . .
Human subjects. Uninfected individuals were defined.as healthy individuals with a negative tuberculin skin test (TST) and no know risk factors=for infection with Mtb. Individuals with LTBI were defined as healthy persons with a positive TST and no symptoins and signs of active TB. In all active TB cases, pulmonary TB was diagnosed by the TB Controller of the county and confirmed by positive sputurn culture for Mycobacterium tuberculosis. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood obtained by venipuncture or apheresis.
Media and Reagents. Culture medium consisted bf RPMI 1640 supplemented with 10% Fetal.Bovine Sera (FBS; Bio Whittaker), 5 X 10'51VI 2 ME
(Sigma-Aldrich), and 2 mM glutamine (GIBCO BRL). For the growth and assay of =
Mtb-reactive T cell clones, RPMI 1640 was supplemented with 10% human serum.
. Mtb'strain H37Rv was obtained from the American Type Culture Collection (Rockville, MD) and prepared as previously described (Lewinsohn et al.;
Jlmmunol 165:925-930, 2000). Peptides were synthesized by Geriemed Synthesis, Inc, (San Francisco, CA). Syrithetic peptide pools consisted of 15-iners overlapping.by =11 amino acids (aa) representing Mtb proteins demonstrated to be 'potent CD4 antigens.
Peptide pools representing CFP-10 (Berthet et al., Microbiology 144:3195-3203, 1998; Dillon et a1., JClfn Microbiol 38:3285-3290, 2000), ESAT-6'(Sorenson et al.,.
=Irifect Immun 63:1710-1717, 1995), Mtb39a,(two= pools, A &B, reference) (Dillon et al., Infect Immun 67:2941-2950, 1999), Mtb8.4 (Coler et= al., J Irnmunol 161:2356-2364, 1998), Mtb 9.9 (Alderson et al., JExp Med 191:551-560, 2000), (Coler et al., Jlmmunol 161:2356-2364; 1998), Mtb 9.9 (Alderson et al., JExp Med 191:551-560, 2000),. EsxG (Rosenkrands et al., Electrophoresis 21:3740-3756, 2002),19kDa antigen (Collins et al. JGen Microbio1136:1429=4436, 1990), antigen 85b (.
Borremans et al., Infect Immun 57:3123-3130, 1989) (two pools, A & B, reference) =.
were synthesized. Peptides wei=e resuspended in.DMSO and=up to 50 peptides were combined into one pool such that each peptide.in the pool'was at a concentration of =1mg/ml. 'Peptide pools were stored at -80 C.
Cell Lines and T Cell Clones. EBV-transformed B cell lines, LCL, were. =
either generated using supernatants from the cell'line 9B5-8 (American Type Culture Collecti`on) or obtained from the National Marrow Donor Program (NMDP;
Minneapolis, MN). LCL were maintained by-continuous passage as previously =described ( Heinzel et al., JExp Med 196:1473-1481, 2002). Mtb-specific T
cell clones were isolated from. iindividuals with LTBI or active tuberculosis, using Mtb-infected DCs.as APCs and limiting dilution cloning methodology as previously described (Lewinsohn et al., Jlmmunol 165:925-930, 2000). Briefly, CD8+ T
cells' were.isolated from PBMC using negative selection using CD4 antibody-coated beads and then positive selection using CD8 antibody-coated magnetic =beads per the manufacturer's.instructions (Miltenyi Biotec, Auburn CA) or via flow cytometry.
'In this case, CD4-PE (BD B.iosciences cat # 555347) negative,.CD8-AP.C (BD =
Biosciences, cat# 555369) positive cells (purity >99 !0) were sorted on a Becton Dickenson LSR'II. T cells were'seeded at various concentrations iin the presence of a 1 X 105. irradiated autologous Mtb'-infected DC, generated as described below, and rIL-2 (5 ng/ml) in cell culture media consisting of 200 l of RPMI 1640 supplemented with 10%. human sera. Wells exhibiting growth between 10 - 14 days, were assessed for Mtb specificity using ELISPOT and Mtb-infected.DC as a : -68-source of APCs. T cells, retaining=Mtb specificity were furtl.iei phenotyped for a(3 T
cell receptor expression and CD8 expression by FACS and expanded as desci'ibed' below. V(3 usage was determined'using theIOTest Beta Mark Kit from Beckman Coulter.
5= Expa'nsion of.T cell clones. .To expand the CD8+ T cell clones; a rapid expansion protocol using anti-CD3 mAb'stimulation was used'as described previously (Heinzel et al., JExp Med 196:1473-14 81, 2002):
Generation and Infeetion'ofPeripheral Blood DCs. Monocyte-derived DCs were prepared (Heinzel et al., supra; Romani et al., JExp Med 180:83-93 1994).
.10 To gerierate Mtb-infected DC, cells (1 X 106) were cultured overnight in the presence of Mtb (multiplicity of infection [MOI] = 50:1). After _18 hours, the cells were harvested and resuspended in RPMI/10% human serum.
tIIHC binding assays. The MHC-peptide binding assay utilized measures the ability of peptide -ligands to inhibit the binding of a radiolabeled peptide to purified 15. MHC molecules, and has been described in d'etail elsewhere ( Sidney et al., 1999.
UNIT 18.3 Measurement of MHC/peptide iiiteractions by gel filtration. In Current Protocols in Immunology. Coligan et al., eds., John Wiley & Sons, Inc., 1996).
Briefly; purified MHC molecules, test peptides, and a radiolabeled probe peptide were incubated at room temperature in the presence of human B2-microglobulin and 20 .a. cocktail of protease inhibitors. After a two-.day incubation, binding of the radiolabeled peptide to the corresponding MHC class I molecule was determined by capturing MHC/peptide complexes oii W6/32, antibody (anti-HLA A. B, and C) or B123.2 (anti-HLA B, C and some A) coated plates, and bound counts per m'inute (cpm) were-measured'using a microsciintillation counter. For competition.assays, the =25 - concentration of peptide yielding 50% inhibition of the binding of the radiolabeled peptide was calculated. Peptides were typically tested at six different concentrations co'vering a 100,000-fold dose range, and in three or more independent assays.
Undei the conditions utilized, where [label]<[MHC] and IC50 _ [MHC], the measured values are reasonable approximations of the true Kd values. =
30. IFN-yELISPOT assay. The IFN-y ELISPOT assay was performed as described previously (Beckrnan et al., Jlmmunol 157:2795-2803, 1996). For determination of ex vivo frequencies of CD4+ or CD8+ T'cells responding to Mtb -69- .
infection or Mtb aritigens, CD4+ or CD8+T-cells- were positively selected from =
PBMC' using magnetic -beads (Miltenyi Biotec, Auburn CA) :as a: source of =:
.responder T cells and tested iin duplicate'at four different cell concentrations.
Autologous DC (20,000 cells/vvell) were used as=APC and DC were either infected with Mtb or pulsed with peptide pools (5 g/ml, final concentration of each peptide) aind then added to the assay. For assays using T cell. clones, T cells (1000 or 5000 cells/well) were iricubated with autologous LCL (20,000 aells/'well) in the presence =
or absence of antigen. , =
Data analysis: To deterrnine the ex vivo frequency of antigen-specific T.
cells, the average number of spots per well for each duplicate was plotted against the number of responder cells per well. Linear regression analysis was used to determine the slope of the line, which. represents the frequency of antigen-speoific T
cells. The assay is considered positive, i.e. reflecting the presence of a primed T cell response, if the binomial probability (Lewinshon et al., Microbes Infect

8:2587-2598, 2006) for the number of spots is significantly different by experimental and control assays. To determine differences in ex vivo T cell frequencies between groups, Wilcoxon/Kruskal-Wallis analysis was used.. = .--Example 2 ' .20. Defining Immuriodominant Mtb-Specific CD8+ Antigens To define immunodominant Mtb-specific CD8+ antigens, and to deteimine whether or not these responses result from infection with Mtb, CD8+= T cells were used from donors uninfected, with LTBI, or actively infected with Mtb.
Responses.
=were determined either directly ex vivo, or.using CD8+ T cell clones obtained by - limiting dilution cloning on Mtb-infected autologous DC ( Lewinsohn et al:, J
Immunol 165:925-930, 2000). As much is known about dominant CD4+ =Mtb antigeris, a panel of these commonly recognized antigens was selected for further evaluation. -These were: Mtb39, CFP10, and Mtb8.4, Mtb9.9, ESAT-6, Ag85b, 19kDa, and EsxG. To avoid bias'introduced by usirig peptides of predicte.d HLA-binding specificity, we synthesized overlapping peptides (.15 aa, overlap 11 aa) to represent the proteins of interest (Lewinshon et al., J Immunol 166:439-446, 2001).

-70- =
To_accurately determine the ex vivo' effector cell frequencies of CD8+ T
cells, linear regression analysis was used. As shown, in.Fig. 1, rimagnetic bead purified CD8+ T cells were tested against peptide pulsed DC over'a iange of CD8'~'T
cell.
numbers in an IFN-y ELISPOT assay. A positive assay' was determined as described below and if positive, the antigen= specific frequency was =determined using linear regression.

Subjects uninfected (n = 14); those with LTBI (n = 20) and tliose with active TB (n = 12) were evaluated for CD8+ responses to a panel of Mtb CD4+ T cell antigens, as well as to Mtb-infected DC. All subjects tested had robust CD8+ T
cell responses to Mtb-infected DC and were of greater magnitude in individuals with active TB. than in those with=LTBI (p = 0.01; Fig..2, Table I). However, CD8+T
cell responses to the panel of Mtb antigens were found almost exclusively in those infected with Mtb in that statistically significarit differences between unirifected and Mtb-infected individuals were noted for seven of ten antigens for both the rinagnitude of the response (Fig. 2) and the proportion of positive assays (Table I).
Table I. CDB+.T cell responses to known TB antigen's. ' =
Antigen Mtb Infected Mtb Uninfected P value (2 tail fishers) # positivea / # tested (%) # positivea / # tested (%) Mtb DC ' 17/17 (100) 11/11 (100) Mtb39'Pool A 13/30 (43) 0/14 (0) 0.003 Mtb 39 Pool B 10/30(33) 0/14(0) 0.01 CFPIO 14/30 (47) . 1N (7) 0.02 Mtb 8.4 ~ = 13/30 (43) , .0/14(0) 0.003 Mtb 9.9 10/25(40) 0) = 1/14 (7) : 0.06 .=
ESAT 6 12125 (48) , .0/14 (0) ' = 0.003 Ag85b Pool A 5/22 (23) 1/14(7) = 0:37 =
Ag85b Pool B 4/22(18) = =0/14 (0) = . 0:14 ' =
19 kd = . 6/22(27) 1/12(8) =0.38 EsxG = ' 9/22(41) ' 0/14 (0) 0.006 Positive assay defined in text. =

However differences =in CD8} T cell responses between individuals with =active TB and LTBI were not statistically different. While.strong CD8+ T cell responses were observed against many of the antigens tested, it is equally notable that several subjects with strong Mtb directed CD8* T cell responses'did,not'have demonstrable. responses to many' of the antigens tested.

These ex vivo frequency data' d'emonstrate the presence of high-frequency responses, to a number:of known Mtb antigens, but do nQt shed light'on the restricting allele, minimal epitope, or dominance hierarchy within the gene of interest. To address this question," limiting dilution cloning of human CD"8+
T cells using Mtb-infected DC was performed (see Lewinsohn et al., Jlmmunol 166:439-446, 2001), and panels of both-classically and iion-classically'HLA-restricted CD8t Tcell clones weire generated. Using peptide pools representing known CD4 antigens, the antigenic specificity of the HLA-la restricted clones can be =defined in.
more than half of the clones (Table II).
Table U. Many CD8+ T cell clones rccognize known CD4+ T cell antigens ' Donor Tb Status HLA-Ia Clones Antigen Identified # Distinct' # Distinct (#)a (W Antigens (#)` Epifopes (#)d D431 Active TB 1 0 0 0 D432 ' Active TB 14 4 2 . 2 D466 Active TB 11 = 10 1 = " 2 D571 Active TB 7 = . =7 . 1 1 D480 Active TB 6 6 = 1 1 D481 Active TB 11 = 11 . = "= 1 . 1 D426 LTBI 1 0 0 . 0 D443 == LTBI I . 1 I 1 D454 LT`BI 2 " "= 2 2 2=
D504 LTBI . 7= 1 1 1 Totals . 61 = 42 10 ' 11 e Number of clones derived from donor. "

b Number of clones for which" cognate antigen was identified. Total number of distinct antigens identifed from the clone set. d Total number of distinct epitopes identified from the clone set.

. =. =
This approach is demonstrated in detail for a single representative clone, ' D466 D6, derived from a siubject with active TB. As shown in Fig. 3A, testing the clone against autblogous DC pulsed with a panel of peptide pools unambiguously 'defined the antigenic specificity as CFP10. The clone was theri tested against each of the 15-mer peptides that comprise the CFP 10 pool, revealing that the epitope "was contained within CFP101-15 (Fig. 3B). Each possible 8' aa, '9 aa, 10 aa, and 11= aa peptide was then synthesized and tested for reactivity, revealing antigenic activity 'between aa 2-11 (Fig. 3C). Similarly, each clone was tested against lymphoblastoid cell lines (LCL) sharing at least one HLA-type with the donor (Fig: 3D).

Autologous LCL and IHW 9058 LCL, which share B4501' and C1601, pre'sent the epitope to the clone, identifying both B4501 and C1601 as possible resti-icting .
alleles. However, C1601+ D433 LCL.do not preserit=the epitope, el'iminating as a candidate restricting allele. Therefore D466 D6 is:restricted by. HLA-B4501.
As demonstrated in Fig. 4, by testing each plausible epitope= over a broad range of . concerttrations, the minimal epitope was defined as CFP 102-IO :for D466 D6.
Experimental data supporting the assignment'of the minimal epitope is provided for each cloile in the supplemental Figure. A summary of the antigenic specificity, minimal'epitope, and HLA-restricting allele is presented in Table ITI.
Unexpectedly, . all=but one of the T cell clones were restricted-by HLA-B alleles.
Furthermore,'a' minority of those observed were 9 aa in length. ' Table III. Summary of Epitopes Identified ' Clone Gene Accession HLA- ' Epitope Epitope Sequence # MHC V beta Nuniber Restrict Locat'n 38}Q ID NOs: 26- '= SFUb gind.Aff. region.
Allele D160 I-1B .(0) - CFPIO Rv3874 =B44 2-11 ' AEMKTDAATL .'360 38 D1601-6F (0) CFPIO Rv3874 B14 85-94 RADEEQQQAL 120 NA
D432 H12 (2) CFPIO Rv3874 B3514 49-58 TAAQAAWRF 258 2011` 5.3 D466 AIO (10) CFPIO Rv3874 = B4501 2-9- . AEMKTDAA 2458 48 - IND
D466 D6 (1) CFPIO Rv3874= B4501 2-12 = AEMKTDAATLA- 1993 6.2 22 -D481 C10 (10) CFPIO Rv3874 B1502 75-83 . NIRQAG.VQY 1715 = 14r 9 D48.1 C11 (1) '='CFP10 Rv3874= B1502 75-83 ' NIRQAGVQY 1715 14r = 13.6 .
D480 F6 (6) CFPIO. Rv3874 B0801 .= 3-1 1 EMKTDAATI. 387 79 13.1 D571.B12 (3) . CFPIO = Rv3874 = B4402 2-11 AEMKTDAATL 31 . 38 = , IND =
D571 E9(4) CFP10 =Rv3874 B4402 2-11 ' =AEMK'TDAATL= ' 31 38 14 D504 E4 (1) Mtb9.8 Rv0287' A0201 3-11 ..= LLDAHIPQL =<10 0.39 8 D454 B10 (1) Mtb9.8 Rv0287 B0801 53-61 AAHARFVAA 88 .6.22. IND
D454 H1-2 (1) Mtb8.4 Rv1174c .B1501 5-I5 AVINITCNYGQ 24.. 10 7.1 D432 A3 (2) Mtb 8.4 Rv1174c B3514 32-40 ASPVAQSYL 210 127c 14 D443H9(1) Ag85B Rv1886c TBD 144-153 ELPQWLSANR <10 NA . 22 Nuniber of sister clones is in parentheses.
b# of SFU /250,000 CD8'' T cells is shown. -`IC50 in nm is shown. _ Published previously J. Immunol. 2001 Jan 1;166(I):439-46. .
`Ivleasured binding affinity to B3501 is shown..
== =
' = .
rMeasured binding affinity to B1501 is shown.
NA = Not Available. = , ' IND = Indetenninate TBD = To be done.

-73- '.
Because eaeh -of the individual CIJ8+ T cell c1'ones were derived based an .
growth of Mtb- infected DC, it was determined whether or not the antigen and =
epitopes identified reflected.immunodorninant epitopes ex vivo. Tw&
independent.
=approaches were pursued, the first to determine if the 'r.esponse. was, present at high frequency, and the second to determine what proportion of the total response to the aritigeri is'constituted by the epitope. To determine the ex-vivo effector cell ' frequency, as clescribed in Fig: 1, each epitope was tested tzsing autcilogous DC and magnetic bead purified CD8+ T cells derived from the=donor from whom the T
cell clones was isolated. A summary of the effector cell frequencies is presented in ' Table III. For the majority, the epitopes reflect high frequency responses, and thus could be considered. a response that has been primed by exposure to Mtb.
Notably, T cell clones isolated from four donors recognized CFPIO. To determine if the epitopes defined reflected a substantial proportion of the total response to' the antigen of interest, magnetic bead purified CD8+ T cells from three donors with sufficient available peripheral blood mononuclear cells (PBMC) were tested for reactivity to each individual 15-mer peptide, the peptide pool, and peptide representing the minimal epitope. As is deinonsrrated iri Fig. 5, the-ex vivo frequencies to the minimal epitope; 15-rrier peptide(s) containing the minimal epitope, and peptide pool were remarkably concordant. 'These- data suggested that 'for each donor a dominance hierarchy has'been clearly established, and is reflected in the original clones. Finally, as is noted in :Table III, daughter clones of identical specificity were fre.quently identified, a result that would be predicted based on'an ' immundominance hierarchy.. TCR V beta staining was used to confirm the clonal relationship between daixghter clones.. Interestingly, in two. cases, the identical minimal epitope and HLA-restrictioin was represented by two distinct clones*(Table III). - = - . .
Because much work on human CD8+ T cell responses to Mtb has relied upon the use of HLA-prediction algorithms, as each epitope was defined we -asked whether or not the epitopes would have been predicted by these approaches.
Many 'of-these epitopes were not ranked strongly. This might-highlight the limitations of .
those algorithms at the time they were used. To address this qilestion experirnientally, the IC50 for each peptide that had been synthesized in the course of definition of the minimal epitope was determined against. a panel of human HLA
molecules. Shown in Table III is the IC50 for the minimal epitope with the cognate restricting allele. =The data demonstrated that the T cell epitopes bound avidly to HLA, and show a high degree of concordance betvveen the T cell epitope data'and .5 HLA-binding data. ' The data demonstrated that CD8+ T cell responses, are present- in persons infected with Mtb at frequencies_ that are comparable to that seeri following many common viral infections such as vaccinia, influeiiza, and CMV. All.but one of the epitopes that were mapped were restricted by HLA-B molecules. The data suggest that by using a T cell driven approach to epitope identification, dominant epitopes can be defined in humans infected with Mtb.

Example 3 Screening of T cell clones against a genomic peptide library.
The classically-restricted and non-classically-restricted T cell clones (see Table II.-above) that did not recognize one of the known Mtb aritigen peptide pools (Rv3875, Rv3874, Rv1886c, Rv0287, Rv3763, Rvl 174c, Rv1196, Rv1793, -Rv2346c, Rv1037c, Rv3619c and Rvl'198) were= screened against a genomic peptide library. This peptide library represents 389 genes, representing roughly.10%
of the Mtb genome. The peptides are 15mers overlapping by..11 for each gene product.
50nmol of each peptide was synthesized individually and then pooled into 777 pools of 50 peptides in a 96 well format (nirie plates). Five blank wells and one well of an irrelevant peptide pool, SIV gag, were'included on each of the niiie plates.
To screen the clones against the genorriic peptide library, the clones 'are first expanded and tested against Mtb-infected DCs to ensure that each clone from: this particular expansion yields a robust Mtb-specific signal in the ELISPOT assay.- Then up to six T cell clones are pooled. For the screen, T cell clones (5,000 cells/well of each clone),=autologous DCs (20,000 cells/well), IL-2 (0.5ng/ml)'and the peptide=pools (5ug/ml, individual peptides) were incubated overnight at 37C in the ELISPOT =
assay. Only one technical replicate is done per pool because 5000 T cell clones per well with a peptide antigen produced= an overwhelmingly positive= resporise, resulting-in a definitive resiilt. Six classical clones from D504 were screened against the genomic peptide library, leading to.the discovery of a,new epitope. This epitope was from a:family of four proteiils that includes.EsxJ, EsxW, EsxK and EsxP.=
These proteins share 98% homology and differ at only-3 amino"acids. There=is a fifth member of this family, EsxM (Rv1792); that was not=included.in the genomic peptide library.
The clones, were screened against the individtial fifteen-mers for these peptide pools. All.six classical clones recognized EsxJ 21-35. This is a region of ' EsxJ that is identical to the other four niembeis bf this faniily. Next, 9, 10 .and=
l lmer peptides were made from this 15mer and screened against each clone. The = minimal epitope was determined to be EsxJ 24-34. In addition, the HLA
restriction was found to be B5701.

Example 4 Additional.Screening of T cell clones against a genomic peptide library . ' Eleven classical clones from D432B were screened against the genornic peptide library described. above. The= antigen was determined- for two clones, which led to the identification of two novel epitopes, PE PGRS4247-55 and PE953-67.=
The *
minimal epitope for one clone was determined to=be PE_PGRS4247-55 and the HLA
restriction'was found to be B3514. The minimal epitope for the other clone is not yet determined, but is contained in the 15mer PE953_67. The HLA'restriction for this clone'was found to be B3905.

Table IV. Detail of Novel Epitopes from Genomic Peptide Library Screexis.
Clone Gene Accession Epitope Epitope #SFU/ MHC- LYIHC' TCR V
Number ' Location = 250,000 Restriction Binding beta CD8+ . Affinity region T-cells (IC50 nm) D504 EsxJ* ' Rv1038c 24-34 QTVEDE- 84 B5701 TBD Indeter-F9 (6) = = SEQ ID NO: ARRMW minate D432 ' PE9 = Rv1088 . 53-67 RLFNAN- TBD ' B3905 TBD = 8 D8 ' SEQ ID NO: AEEYHA-(1) .7 LSA
D432 PE PGR Rv2487c 47-55" VSAAIAG- TBD B3514 TBD 7.1 H8 S42 - SEQ ID NO: LF
(1) . 8 Number of clones.recognizing epitope from each donor in parentheses. *This i's a family of proteins that have almost identical sequences. The family consists of Rv1038c, Rv1197, Rv2347, Rv3620c.
-= -76- ' Table V. Suinmair"y of Completed Clone Screens. "
= . #
classical # Non- .# classical clones # Classical Clas'sical # positive # of clories epitope TB available available wells in confirmed # no'vet epitope NOT
Donor Status scrccned screened screen hits e ito es identified identified 426 PPD+ 1 1 4(4) 1 0 0 0`
431 . Active 1 1' 1 1 l** =- 0 0 0 1 432 Active 11 11 14 (7) 11 3. 2 '3 = 8 454 PPD+ 1* (0) 6(4) 0 .0 0 = 0. 0' 466 Active ' 1 1 = 4(4) 1 0 . 0 0' 1 504 PPD+ 6(6) 9 9 5 .4- = 1 '.6' = 0=
21 (20). 38(29) 18 7 3 9 11 * The classical clone.from D454 did not recognize Mtb upon re-expansion and was not screened against library.
** The classical clones from 426 and 431 wero screened together, so there was one positive well between both clones.

' ExampCe 5 Screening of ex vivo CD8+ T-cells against a genomic peptide lifarar,y CD8+ T-cells from a LTBI donor, D610 (SE Asian) were screened against the =genomic peptide library described above. Each plate of the genomic peptide library' was screened in duplicate, for a total of 18 ELISPOT plates per screen. CD8+
T-cells wei=e prepared from cryopreserved PBMC by CD8+ selection using magnetic bead separations. Resulting,cell populations 'contained >96 f CD8"T cells.
CD8+ T
cells (250,000 cells/well), autologous DCs (20,000 cells/well), aild IL-2 (0.5ng/ml) were added to peptide (final5ug/ml, individual peptides) in the ELTSPOT
plates.
Five media control wells are inclilded on each plate. For each plate, fhe.mean of these five wells was subtracted from each well of that plate to normalize between plates. Each technical replicate on each plate was then scored. A well was scored positive if the spot forining units (SFU), less the mean of the media wells, .
was greater than or equal to ten and the SFU was greater than or.equal to twice the mean of the media. (Hudgens et al., J. Immunol. Methods 288: 19-34, 2004). This donor responded to the.four,peptide wells containing EsxJ, EsxW, EsxK and EsxP. CD8+
T-cells were then screened against each 15mer from these peptide pools aind found to respond. only to EsxJ 21-35,.the same region of EsxJ, EsxW, =EsxK and EsxP
that==
'is Aescribed in example 3 above: = =
Seven additional donors were screened against the genomic peptide library..
The top 10 i=esponses are detailed in"Table 7. The four peptide pools highlighted in : yellow contain peptides from= only one.gene. ,These four= genes c'ontain four novel epitopes.

Table 7. Top 10 Responses from Peptide Pool Screens of Seven Donors. Spot.
Forming Units are for 250,000 CD8+ T-cells.

Peptide Average .'Pool Donor SFU 'RvNumbers'Re resented in Wells = =Runctional Cate o cell wall"and cell C09 1 D560 208.2 Rv 1860 50 :' =' proecsses C12 4 D545 .156.4 Rv0468 27 : Rv0456c 23 =' : = = lipid metabolism cell wall and cell A04 3 D454 = 136 Rv0284 ~17 : Rv0288 1 I : Rv0287 22 processes cell wall and=cell B10 3 D560 112.3 Rv1273c 50 : ' processes E04 4= D560 78.2 Rv0152c 40 : Rv0151c 10 : PE/PPE
G 12 8 D560 77.4 Rv3478 18 : Rv3507 32. : PE/PPE.
.E07 D525 76.8 Rv0159c 50 : PE/PPE
A10 8 D560 70.4 Rv3136 47 : R0144c 3: PE/PPE
E11 8 D560 66.4 Rv3350c 50 : = PE/PPE
E09 9 D545 60.2 Rv1404 13 : Rv2711 37 : = re lato roteins Example 6 ' . . Animal models In tuberculosis research, the mouse model has been used extensively to model various aspects of the disease. Mice can be infected by a variety of routes, including intravenous, intraperitoneal and tracheal. One route is aerosolization of =
the organism for respiratory infection. The mice are exposed to the aerosol in :a chamber (wither whole body or nose only infection). The dose of invention can be varied by manipulating the concentration of Mtb in the nebulizer or time of = exposure. A low dose infection, such as about 50 colony forming units (CFU) via aerosol reulsts in a slow and steady increase in bacterial numbers in the lungs, generally reaching a peak in four weeks, which coincides ~with the peak number ofT' cells in the lungs: The initial period is considered.the acute stage of irifection.
=Folliwng infection, there is a dissemination of bacteria to the mediastinal lymph nodes. T cell priming is generally detectable between two and three weeks.
After about four weeks the= bacterial numbers stabilize, and there is a slow progressive pathologic response. This system is of-use for modeling active infection. :
The ability of a composition of interest to prevent infection in an animal mo.del can be evaluated using the methods described herein. The effectiveness, of .

-78- =. .

the cotnposition of iriterst can be monitored by measuring`tlie =T cell response, such.
as the number of CD8+ or CD4+==T cells responding to an.Mtb polypeptide in a biological sample. For these assays T cells with one are. contacted with at least one Mycobacterium polypeptides, and an antigern presentirig cell presenting the one. or 5. more. Mycobacteriurim polypeptides. The Mycobacterium.polypeptides include the amino= acid :sequence set forth as (a) one of the. am.ino acid sequences 'set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,'SEQ ID NO: 5, SEQ
ID NO: 6,= SEQ ID NO: 7, SEQ ID NO: 8, =SEQ ID NO: 9, SEQ ID NO: 10,'SEQ I1D
NO: 11 or SEQID NO: 12; or (b) at least nine to twenty consecutive amino acids of at least one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO:
2, SEQ ID NO: 3,= SEQ ID NO:. 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ' ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ I'D NO: 10, SEQ ID.NO: 11 or.=SEQ
ID NO:'=12, wherein the nine to twenty consecutive amino acids specifically bind major histocompatibility complex (MHC) class I. 'It is determined if the determining _ if the T cells specifically recognize the Mycobacterium polypeptide. An.
increase in the number of T cells that specifically recognize the Mtb polypeptide indicates that the composition is effective. = . ' ' Exemplary anirrial models aie described below (see also Repique et al., Infec. Inimun. 70: 3318-3323, 2002, incorporated herein by reference- for an additioinal protocol):

A. Short Term Mouse Model:. = .

C57BL/6= mice are vaccinated with a composition accordiing to the= =
appropriate protocol and then rested for 4 to 6 weeks. Immunized mice are infected with a low dose aerosol 50-100 CFU) of virulent M. tuberculosis and protection i's evaluated by assessing the number of viable bacilli 30 days post challenge.; .
Viable counts are performed on the'lung and spleen of mice.by homogenizing the organs and plating serial 10-fold dilutions on 7H11 agar plates.
Plates are incubated= for up to 21 days and the number of colony, forming units per organ determined. . , BCG vaccinated mice have approximately 1Log10 protection in their lung and spleenwhen.compared to PBS-treated mice.

-79-=
A biological sample is obtained prior to the= administration of the compositiori of interest and after administration of the composition. of interest.
Alternatively, biological samples are obtained from vehicle treatedanimals and fi=om an irnals treated with the composition of interst. An increase in the number of T'cells that biiid ari Mtb polypeptid'e as disclo'sed hereiri 'indicates 1lie composition is -effective.
B. Short term guinea pig moa?el . .
Out-bred-Hartley guinea pigs are vaccinated with a composition including one or more Mtb polypeptide, or a polynucleotide encoding these one or more polypeptides and then rested for 8 to 10 weeks. Immunized guinea pigs are infected -with a low dbse aerosol (10-30 CFU) of virulent M tuberculosis.and protection is evaluated, by assessing the number of viable. bacilli 30 days post challenge.

Viable counts are performed on the lung and spleen of guinea pigs by15 homogenizing the organs and plating serial 10-fold dilutions on 7H11 agar plates..
Plates.are incubated for up to 21 days and the number of colony forming units per organ'determined. Lung.and spleen segments'are also taken for histological analyses. = :
BCG.vaccinated'guinea pigs have approximately. 22-3Logio protection in their lurig and spleen when compared to PBS-treated guinea pigs. In addition, BCG.
vaccinated guinea pigs have well defined granulomas when'compared to unvaccinated animals. . A biological sample is obtained prior to the administration of the composition of interest and after administration of the composition of interest.
Alternatively, biological samples are obtained from vehicle treated animals and from =
animals treated with the composition of interst. An increase in the number of T cells that bind an Mtb polypeptide as disclosed herein indicates the composition is' effective. -C. Long term.guinea pig model . =. .
The guinea pig model is similar to the moiuse rnodel, but the experiments are =
open-ended survival type and can last for as long as 2 years. = Guinea pigs develop : -80=. =

`classical' granulomas similar to humans with active tuberculosis (TB),=and as'lung tissue necrosis progresses, they begin to lose weight and die of TB similar to humans. The riumber of colony forming uriits iri the lungs aiid spleen can be assessed. Histological examination=can'also be=perforrned to determine the degree of liung involvemeint=and tissue destruction. After low-dose aerosol exposure in the guinea pig the nurnber of organisms increases progressively during the =first three weeks'and then plateaus into a chronic state. During the later stages of infection there is increased bacterial load iri the lung=and this is associated- with a worsening:
pathological condition. Without treatment, there is a concomitant rise in both and CD8 T celTs in the lungs of infected guinea pigs.
Out-bred Hartley guinea pigs =are -vaccinated with the experimental vaccine according to the appropriate protocol and then rested for 8 to 10 weeks.
Immunized' = guinea'pigs are thein infected with a low dose aerosol (10-30 CFU) of virulent M.
tuberculosis. Guinea pigs are =weighedweekly and monitored daily for signs of disease (such as increased respiration and.failure =to thrive). Unvaccinated 'guinea.
pigs succumb to infection from 20 to 25 weeks post challenge, while BCG
vaccinated guinea pigs survive for 50 to 55 weeks post challenge.
At necropsy, the lung and spleen are assessed for= the numbei of CFU and the extent of pathology. The.relative protection of the experimental:composition is '20 compared to BCG vaccinated animals.
A biological =sample is obtained prior to the administration of the.
composition of.interest and after administration of the composition of iriterest.
Alternatively, biological samples are obtained from vehicle treated animals and from animals treated with the composition of interst. An increase in the number of T cells = that bind an Mtb polypeptide as disclosed herein indicates the composition is effective. It will be apparent that the precise details -of the methods or compositions described may be varied or-modified without departing from the spirit of the. -described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below. ' = :

Claims (49)

CLAIMS.

1. A method for detecting Mycobacterium tuberculosis in a subject, comprising contacting a biological sample from the subject comprising T cells with one or more Mycobacterium polypeptides, and an antigen presenting cell presenting the one or more Mycobacterium polypeptides wherein the one or more Mycobacterium polypeptides comprises an amino acid sequence set forth as (a) one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO:
2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQID NO: 12;
or (b) at least nine to twenty consecutive amino acids of at least one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 7, SEQ
ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, wherein the nine to twenty consecutive amino acids specifically bind major histocompatibility complex (MHC) class I; and determining if the T cells specifically recognize the Mycobacterium polypeptide, wherein the presence of T cells that specifically recognize the Mycobacterium polypeptide detects Mycobacterium tuberculosis in the subject.

2. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1.

3. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 1 that specifically binds MHC class I or a polynucleotide encoding the polypeptide.

4. The method of claim 3, wherein the Mycobacterium polypeptide comprises the amino acid sequence QTVEDEARRMW (amino acids 24 to 34 of SEQ ID NO: 1).

5. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 2.

6. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 2 that specifically bind MHC class I.

7. The method of claim 6, wherein the Mycobacterium polypeptide comprises the amino acid sequence VSAAIAGLF (amino acids 47 to 55 of SEQ ID
NO: 2).

8. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 3.

9. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 3 that specifically binds MHC class I.

10. The method of claim 9, wherein the Mycobacterium polypeptide comprises amino acids 53 to 67 of SEQ ID NO: 3.

11. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 4.

12. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 4 that specifically bind MHC class I.

13. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 5.

14. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 5 that specifically bind MHC class I.

15. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 6.

16. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 6 that specifically bind MHC class I.

17. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 7.

18. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 7 that specifically bind MHC class I.

19. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 8.

20. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 8 that specifically bind MHC class I.

21. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 9.

22. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 9 that specifically bind MHC class I.

23. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 10.

24. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 10 that specifically bind MHC class I.

25. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 11.

26. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 11 that specifically bind MHC class I.

27. The method of claim 1, wherein the Mycobacterium polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 12.

28. The method of claim 1, wherein the Mycobacterium polypeptide comprises at least nine consecutive amino acids of SEQ ID NO: 12 that specifically bind MHC class I.

29. The method of any one of claims 1-28, wherein the T cells are CD8+ T
cells.

30. The method of any one of claims 1-29 in which determining if the CD8+T cells specifically recognize the Mycobacterium polypeptide is determined by measuring secretion of a cytokine from the CD8+ T cells.

31. The method according to claim 30, wherein the cytokine is interferon (IFN)-.gamma..

32. The method of claim 31; wherein measuring secretion of IFN-.gamma. is determined using an antibody that specifically binds IFN-.gamma..

33. The method of any of claims 1-32, wherein the biological sample is blood, isolated peripheral blood mononuclear cells, isolated mononuclear cells.

34. The method of any one of claims 1-32, wherein the T cells are cultured in vitro with the Mycobacterium polypeptide.

35. The method of any one of claims 1-33, wherein the polypeptide is administered to the subject.

36. A method of detecting Mycobacterium tuberculosis in a subject, comprising;
administering to the subject an effective amount of a Mycobacterium polypeptide into the skin of the subject, wherein the Mycobacterium polypeptide comprises an amino acid sequence set forth as (a) one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO:
2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQID NO: 12;
or (b) at least nine to twenty consecutive amino acids of at least one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6; SEQ ID NO: 7, SEQ ID NO: 7, SEQ
ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, wherein the nine to twenty consecutive amino acids specifically bind major histocompatibility complex (MHC)class I; and detecting the presence of CD8+ T cells that specifically recognize the Mycobacterium polypeptide in the subject;

37. The method of claim 36, wherein detecting the presence of CD8+ T cells comprises detecting the presence of CD8+ T cells in vivo.

38. The method of any one of claims 35-37, wherein the detecting the presence of CD8+ T cells comprises detecting a delayed type hypersensitivity reaction.

19. The method of any one of claims 35-38, wherein the Mycobacterium polypeptide is administered intradermally to the subject, and wherein the delayed type hypersensitivity reaction is measure by measuring redness, swelling or induration of the skin.

40. A method of detecting a Mycobacterium tuberculosis infection in a subject, comprising detecting the presence of a Mycobacterium polypeptide or a polynucleotide encoding the polypeptide in a sample from the subject, wherein the Mycobacterium polypeptide comprises an amino acid sequence set forth as one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQID NO: 12.

41. The method of claim 40, comprising determining the presence of the Mycobacterium polypeptide.

42. The method of claim 41, wherein detecting the presence of the Mycobacterium polypeptide comprises the use of an antibody that specifically binds the Mycobacterium polypeptide.

43. The method of claim 40, comprising determining the presence of the Mycobacterium polynucleotide.

44. The method of claim 43, wherein determining the presence of the Mycobacterium polypeptide comprises the use of polymerase chain reaction.

45. The method of claim 40, wherein the biological sample is blood, peripheral blood mononuclear cells; sputum, a lung biopsy, a lymph node biopsy, saliva, cerebral spinal fluid or isolated T cells.

46. A method of detecting T cells expressing CD8 that specifically recognize SEQ ID NO: 1 in a subject, comprising (A) contacting peripheral blood mononuclear cells isolated from the subject with the a reagent comprising (1) a Mycobacterium polypeptide comprising at least nine to twenty consecutive amino acids of at least one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, wherein the nine to twenty consecutive amino acids specifically bind major histocompatibility complex (MHC) class I;
(2) HLA heavy chain polypeptide and .beta.2-microglobulin: and (3) strepavidin, wherein the reagent is labeled or unlabeled; and (B) detecting the presence of the reagent bound to the peripheral blood mononuclear cells, thereby detecting T cells expressing CD8 that specifically bind the Mycobacterium polypeptide.

47. The method of claim 46, further comprising quantitating the number of CD8+ T cells that bind the reagent.

48. The method of claim 46, wherein the reagent is labeled.

49. The method of claim 48, wherein the label is a fluorochrome.